2-substituted benzothiazolyl-3-substituted phenyl-5-substituted sulfonated phenyl-2h-tetrazolium salt, reagent for biological component concentration measurement containing said salt, and biological component concentration measurement method using said salt

ABSTRACT

A 2-substituted benzothiazolyl-3-substituted phenyl-5-substituted sulfonated phenyl-2H-tetrazolium salt represented by the following Formula (1): 
     
       
         
         
             
             
         
       
     
     wherein, R 1  can be a hydrogen atom, a hydroxyl group, a methoxy group, and an ethoxy group; R 2  can be a nitro group, —OR 4 , and a carboxyl group; R 3  is a hydrogen atom, a methyl group, or an ethyl group, while at least one is a methyl group or an ethyl group; R 4  is a methyl group or an ethyl group; m is 1 or 2; n is an integer from 0 to 2; p is 0 or 1; n+p is 1 or greater; q is 1 or 2; when q is 2, the OR 3 &#39;s are disposed adjacently to each other and may form a ring; and X is a hydrogen atom or an alkali metal atom.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a bypass continuation of PCT Application No. PCT/JP2017/031677,filed on Sep. 1, 2017, which claims priority to Japanese Application No.2016-179911, filed on Sep. 14, 2016. The contents of these applicationare hereby incorporated by reference in their entireties.

BACKGROUND

The present disclosure relates to a 2-substitutedbenzothiazolyl-3-substituted phenyl-5-substituted sulfonatedphenyl-2H-tetrazolium salt, a reagent for biological componentconcentration measurement including the salt, and a method for measuringa biological component concentration using the salt.

BACKGROUND ART

In clinical chemical examinations, there are available methods fordetecting and quantitatively determining the amount of a biologicalcomponent included in a body fluid of an organism, such as blood orurine, depending on the amount of a coloring material that is detected,and reagents used for these methods are referred to as indicatorreagents.

For example, JP 8-53444 A discloses a method for quantitativelydetermining a reduced nicotinic acid amide adenine dinucleotide using awater-soluble tetrazolium salt compound having the following structure:

in which R¹ represents a hydrogen atom or a methoxy group; R² representsa hydrogen atom, a carboxyl group, or sulfonic acid.

SUMMARY

Formazan dye produced from the tetrazolium salt of the aforementioned JP8-53444 A is highly water-soluble; however, the color developmentintensity in the wavelength range (600 nm or greater) that does notoverlap with the main absorption band of hemoglobin is not sufficient.For this reason, when the tetrazolium salt of the aforementioned JP8-53444 A is used, sufficient sensitivity cannot be achieved for wholeblood samples.

Therefore, the present disclosure was achieved in view of suchcircumstances, and for a reagent for biological component concentrationmeasurement has a sufficient coloring peak in a wavelength range (600 nmor greater) that does not overlap with the main absorption band ofhemoglobin. Thereby, it is an object of the disclosure to provide ameans capable of quantitatively determining a biological component withsufficient sensitivity even for a whole blood sample.

Another object of this disclosure is to provide a means capable ofmaintaining the water-solubility of a reagent for biological componentconcentration measurement while quantitatively determining a biologicalcomponent stably with sufficient sensitivity even in the case of usingthe whole blood as a sample.

The inventors of the present disclosure conducted a thoroughinvestigation in order to solve the problems described above, and as aresult, the inventors found that the issues described above can beaddressed by a tetrazolium salt having a benzothiazolyl group with amethoxy group or an ethoxy group introduced therein, at the 2-positionof a tetrazole skeleton; a substituted phenyl group at the 3-position ofthe tetrazole skeleton; and a phenyl group having at least one sulfogroup (—SO₃ ⁻), at the 5-position of the tetrazole skeleton. Thus, theinventors have completed the present disclosure.

That is, the above-described object can be achieved by a 2-substitutedbenzothiazolyl-3-substituted phenyl-5-substituted sulfonatedphenyl-2H-tetrazolium salt represented by the following Formula (1):

in which in Formula (1), R¹ represents any one selected from the groupconsisting of a hydrogen atom, a hydroxyl group, a methoxy group, and anethoxy group; R² represents any one selected from the group consistingof a nitro group, —OR⁴, and a carboxyl group (—COO⁻), while multipleR²'s may be identical with or different from each other; R³ represents ahydrogen atom, a methyl group, or an ethyl group, while at least one isa methyl group or an ethyl group; R⁴ represents a methyl group or anethyl group; m represents the number of sulfo groups (—SO₃ ⁻) bonded tothe phenyl group at the 5-position of the tetrazole skeleton, and is 1or 2; n represents the number of R²'s is bonded to the phenyl group atthe 3-position of the tetrazole skeleton, and is an integer from 0 to 2;p represents the number of sulfo groups (—SO₃ ⁻) bonded to the phenylgroup at the 3-position of the tetrazole skeleton, and is 0 or 1; n+p is1 or greater; q is 1 or 2; when q is 2, the OR³'s are disposedadjacently to each other, while in this case, the OR³'s may be bonded toeach other and form a ring; and X represents a hydrogen atom or analkali metal atom.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing a spectrum of a Ni²⁺ chelate compound offormazan produced from tetrazolium compound 1.

FIG. 2 is a graph showing the relationship between the glucoseconcentration with regard to tetrazolium compound 1 and WST-4 and thelight absorbances of the respective formazans produced (for thetetrazolium compound 1, a Ni²⁺ chelate compound of formazan).

FIG. 3 is a diagram showing the stability evaluation results for thetetrazolium compound 1.

FIG. 4 is a schematic diagram illustrating a blood glucose meter sensorused in Evaluation Example 1.

FIG. 5 is a diagram showing the results of the quantity of signal(absorbance) obtainable when a whole blood sample is applied to bloodglucose meter sensors to which tetrazolium compound 1 and WST-4 areapplied.

FIG. 6A is a diagram showing the spectra of a Ni²⁺ chelate compound offormazan produced when whole blood sample is spotted on a blood glucosemeter sensor employing tetrazolium compound 1.

FIG. 6B is a diagram showing the spectra of a Ni²⁺ chelate compound offormazan produced when aqueous glucose solution is spotted on a bloodglucose meter sensor employing tetrazolium compound 1.

FIG. 7A is a diagram showing the relationship between the time takenfrom the initiation of measurement when a whole blood sample is spottedon a blood glucose meter sensor employing tetrazolium compound 1, andthe absorbance of a Ni²⁺ chelate compound of formazan produced at themaximum absorption wavelength.

FIG. 7B is a diagram showing the relationship between the time takenfrom the initiation of measurement when an aqueous glucose solution isspotted on a blood glucose meter sensor employing tetrazolium compound1, and the absorbance of a Ni²⁺ chelate compound of formazan produced atthe maximum absorption wavelength.

FIG. 8A is a graph showing the relationship between the glucoseconcentration obtainable when whole blood sample is spotted on a bloodglucose meter sensor employing tetrazolium compound 1, and theabsorbance of a Ni²⁺ chelate compound of formazan produced.

FIG. 8B is a graph showing the relationship between the glucoseconcentration obtainable when an aqueous glucose solution is spotted ona blood glucose meter sensor employing tetrazolium compound 1, and theabsorbance of a Ni²⁺ chelate compound of formazan produced.

FIG. 9 is a plan view schematically illustrating a blood glucose meter(component measuring apparatus) equipped with a sensor chip according tothe present embodiment.

FIG. 10 is a perspective view illustrating a magnification of the sensorchip and a photometric unit of the apparatus main body of FIG. 9.

FIG. 11 is a lateral view illustrating the sensor chip of FIG. 9.

FIG. 12A is a first plan view illustrating the operation of mounting ofthe sensor chip and the apparatus main body of FIG. 9.

FIG. 12B is a second plan cross-sectional view illustrating theoperation of mounting subsequent to FIG. 12A.

FIG. 13A is a schematic diagram illustrating a blood glucose metersensor used in Evaluation Example 2.

FIG. 13B is a diagram for explaining the length, width, and thickness ofthe inner surface of the blood glucose meter sensor of FIG. 13A.

FIG. 14 is an absorbance spectrum (blank) of a blood glucose metersensor having water spotted thereon.

FIG. 15 shows a differential spectrum (meaning the net quantity of colordevelopment of glucose) obtained by subtracting the absorbance spectrumhaving water spotted thereon (FIG. 14) from the absorbance spectrum ofthe sensor having spotted thereon aqueous glucose solution at a glucoseconcentration of 400 mg/dL.

FIG. 16 is a diagram showing the relationship between the time takenfrom the initiation of measurement at the time of spotting an aqueousglucose solution (800 mg/dL) on blood glucose meter sensors employingvarious tetrazolium compounds, and the absorbance of a Ni²⁺ chelatecompound of formazan produced at the maximum absorption wavelength(assuming the absorbance obtained 15 seconds after the initiation ofreaction as 100%).

FIG. 17 is a diagram showing the relationship between the time takenfrom the initiation of measurement at the time of spotting a whole bloodsample (Ht40, 800 mg/dL) (hematocrit value 40%, glucose concentration800 mg/dL) on blood glucose meter sensor employing various tetrazoliumcompounds, and the absorbance of a Ni²⁺ chelate compound of formazanproduced at the maximum absorption wavelength (assuming the absorbanceobtained 15 seconds after the initiation of reaction as 100%).

FIG. 18 is a graph obtained by plotting the β-glucose concentration(mol/L) on the x-axis, and plotting the absorbance at λmax=635 nm of acolored chelate compound of formazan and Ni²⁺ calculated to a value per1 cm (abs/cm), on the y-axis.

FIG. 19 is a diagram showing the spectra of a Ni²⁺ chelate compound offormazan produced from tetrazolium compounds 1 and 13 to 18. Theabsorbances of the respective compounds at the maximum absorptionwavelength are taken as 100%.

DETAILED DESCRIPTION

According to a first aspect of the present disclosure, a 2-substitutedbenzothiazolyl-3-substituted phenyl-5-substituted sulfonatedphenyl-2H-tetrazolium salt having a structure represented by thefollowing Formula (1):

is provided. According to the present specification, the 2-substitutedbenzothiazolyl-3-substituted phenyl-5-substituted sulfonatedphenyl-2H-tetrazolium salt of Formula (1) described above will be simplyreferred to as “tetrazolium salt of the present disclosure” or“tetrazolium salt”.

A chelate compound formed from a transition metal ion and formazanproduced from the tetrazolium salt of the present disclosure has themaximum absorption wavelength in a wavelength range (600 nm or greater)that does not overlap with the main absorption range of hemoglobin. Forthis reason, the influence of coloring substances existing in the bloodis low, and the measurement error can be reduced. In the presentspecification, the chelate compound of a transition metal ion andformazan produced from the tetrazolium salt of the present disclosure isalso simply referred to as “formazan compound”.

The formazan produced from the tetrazolium salt of the aforementioned JP8-53444 A has maximum absorption at 510 to 550 nm (paragraph [0011]). Infact, in Example 3 of the aforementioned JP 8-53444 A, the NADHconcentration is quantitatively determined by means of the absorbance at550 nm (paragraph [0029], FIG. 2). Meanwhile, in the case of measuringthe concentration of a biological component (for example, glucose) usingwhole blood sample, it is necessary that the measurement wavelength isin a wavelength range that does not overlap with the main absorptionband of hemoglobin. The wavelength for detecting the red blood cellconcentration in the blood is about 510 to 540 nm, and the maximumabsorption wavelength of oxygenated hemoglobin is approximately 550 nm.Therefore, in the case of biological component measurement using thewhole blood as a sample, it is preferable that the maximum absorptionwavelength of formazan be 600 nm or greater. In that case, in theformazan produced from the tetrazolium salt of JP 8-53444 A, theinfluence exerted by blood cells cannot be sufficiently eliminated, andit is difficult to measure the biological component concentration withsatisfactory sensitivity. For this reason, it is necessary to shift themaximum absorption wavelength of the tetrazolium salt of JP 8-53444 Atoward the longer wavelength range side. Generally, when a formazan of acertain kind is subjected to the action of a transition metal ion (forexample, nickel ion or cobalt ion), and thereby a chelate compound isproduced, the maximum absorption wavelength can be shifted toward thelonger wavelength side. However, in Example 3 of JP 8-53444 A, even if atransition metal ion (for example, nickel ion) is added in order toshift the maximum absorption wavelength toward the longer wavelengthside, the formazan does not form a chelate (see Comparative Example 5described below), and when the amount of addition of the transitionmetal ion is increased, a precipitate is formed. Therefore, it is notapt to say that the tetrazolium salt of JP 8-53444 A can be suitablyused as a reagent for a whole blood sample.

In contrast, the formazan compound produced from the tetrazolium salt ofthe present disclosure is such that the maximum absorption wavelength isin a wavelength range (600 nm or greater, particularly 630 nm orgreater) that does not overlap with the absorption band of blood.Therefore, detection noises originating from a biological sample can bereduced by using the tetrazolium salt of the present disclosure. Thatis, a signal related to a biological component can be detected with highsensitivity. The detailed mechanism for providing the above-describedeffects is still not clearly understood; however, the mechanism may beconsidered as follows. In addition, the following mechanism is only aspeculation and is not intended to limit the technical scope of thepresent disclosure.

The inventors of the present disclosure found that since the tetrazoliumsalt of the present disclosure has the benzothiazolyl group at the2-position of the tetrazole skeleton substituted with an alkoxy group,the maximum absorption wavelength of formazan produced from thetetrazolium salt or a chelate compound of formazan and a transitionmetal ion can be shifted toward the longer wavelength side (comparisonbetween Example 2 and Comparative Example 1 that are described below).

Furthermore, by means of the benzothiazolyl group existing at the2-position of the tetrazole ring, the formazan produced from thetetrazolium salt of the present disclosure can efficiently and rapidlyform a chelate compound with a transition metal ion such as Co²⁺ orNi²⁺. This is thought to be due to the nitrogen atom of thebenzothiazolyl group. Here, it is speculated that when thebenzothiazolyl group is substituted with an alkoxy group, since thealkoxy group is an electron-donating group, the electron density of thebenzothiazolyl group increases, and the formation of a chelate compoundof formazan and a transition metal ion is carried out more rapidly.Therefore, it is considered very important to introduce an alkoxy groupinto the benzothiazolyl group existing at the 2-position of thetetrazole ring, in order to shift the maximum absorption wavelengthtoward the longer wavelength side while retaining the chelating abilityof the formazan produced from a tetrazolium salt with a transition metalcompound (comparison between Example 2 and Comparative Example 2described below).

Therefore, when the tetrazolium salt of the present disclosure is used,the maximum absorption wavelength of the formazan compound produced fromthe tetrazolium salt of the present disclosure can be further shifted toa wavelength range (600 nm or greater, particularly 630 nm or greater)that does not overlap with the main absorption band of hemoglobin. Forthis reason, it is made possible to measure a biological componentconcentration with the maximum absorption wavelength existing in awavelength range (600 nm or greater) that does not overlap with the mainabsorption band of hemoglobin, and even a biological componentconcentration in a whole blood sample can be accurately measured byusing the tetrazolium salt of the present disclosure.

Furthermore, in the tetrazolium salt of the present disclosure, thephenyl group at the 5-position of the tetrazole skeleton has one or twosulfo groups (—SO₃ ⁻), and the phenyl group at the 3-position of thetetrazole skeleton has zero or one sulfo group (—SO₃ ⁻). Therefore, thetetrazolium salt has one to three sulfo groups (—SO₃ ⁻) in the compound.Accordingly, the tetrazolium salt is water-soluble. Furthermore, thetetrazolium salt of the present disclosure has excellent stability.

Therefore, a biological component concentration can be measured rapidlywith high sensitivity by using the tetrazolium salt of the presentdisclosure. Furthermore, a biological component concentration can bemeasured with high sensitivity even after a long-term storage, by usingthe tetrazolium salt of the present disclosure.

In the following description, embodiments of the present disclosure willbe explained in detail.

The tetrazolium salt according to an embodiment of the presentdisclosure has a structure represented by the following Formula (1):

In Formula (1), R¹ represents any one selected from the group consistingof a hydrogen atom, a hydroxyl group, a methoxy group, and an ethoxygroup; R² represents any one selected from the group consisting of anitro group, —OR⁴, and a carboxyl group; R³ represents a hydrogen atom,a methyl group, or an ethyl group, while at least one is a methyl groupor an ethyl group; R⁴ represents a methyl group or an ethyl group; mrepresents the number of sulfo groups (—SO₃ ⁻) bonded to the phenylgroup at the 5-position of the tetrazole skeleton, and is 1 or 2; nrepresents the number of R²'s bonded to the phenyl group at the3-position of the tetrazole skeleton, and is an integer from 0 to 2; prepresents the number of sulfite ions (—SO₃ ⁻) bonded to the phenylgroup at the 3-position of the tetrazole skeleton, and is 0 or 1; n+p is1 or greater; q is 1 or 2; when q is 2, the OR³'s are disposedadjacently to each other, while in this case, the OR³'s may be bonded toeach other and form a ring; and X represents a hydrogen atom or analkali metal atom.

The tetrazolium salt according to another embodiment of the presentdisclosure has a structure represented by the following Formula (1′):

In this formula, R¹ represents anyone selected from the group consistingof a hydrogen atom, a hydroxyl group, a methoxy group, and an ethoxygroup; R² represents any one selected from the group consisting of anitro group —OR⁴, and a carboxyl group; R³ and R⁴ each independentlyrepresent a methyl group or an ethyl group; m represents the number ofsulfo groups (—SO₃ ⁻) bonded to the phenyl group at the 5-position ofthe tetrazole skeleton, and is 1 or 2; n represents the number of R²bonded to the phenyl group at the 3-position of the tetrazole skeleton,and is an integer from 0 to 2; p represents the number of sulfo groups(—SO₃ ⁻) bonded to the phenyl group at the 3-position of the tetrazoleskeleton, and is 0 or 1; n+p is 1 or greater; and X represents ahydrogen atom or an alkali metal atom.

In Formula (1), a substituted benzothiazolyl group exists at the2-position of the tetrazole skeleton. In regard to the above-describedFormula (1), since a benzothiazolyl group exists at the 2-position ofthe tetrazole ring, the compound can form a chelate compound with atransition metal compound efficiently and rapidly (the maximumabsorption wavelength of the formazan compound can be shifted to alonger wavelength range). Then, since at least one methoxy group orethoxy group is introduced into the benzothiazolyl group at the2-position of the tetrazole skeleton, the maximum absorption wavelengthat the time of chelation between formazan thus produced and a transitionmetal ion such as Ni²⁺ is further shifted toward the longer wavelengthside (comparison between Example 2 and Comparative Example 1 describedbelow).

Furthermore, q is 1 or 2. Here, in the case of q=1, R³ represents amethyl group or an ethyl group, and from the viewpoint ofwater-solubility, R³ is preferably a methyl group. In a case in which R³is an alkyl group having 3 or more carbon atoms, the tetrazolium saltand formazan produced from the tetrazolium salt have poorwater-solubility, which is not preferable.

In regard to Formula (1), from the viewpoint of the effect of shiftingthe maximum absorption wavelength toward the longer wavelength side atthe time of chelation with a transition metal ion such as Ni²⁺, it ispreferable that at least one of —OR³ of the substituted benzothiazolylgroup existing at the 2-position of the tetrazole skeleton is bonded tothe 6-position of the benzothiazolyl group.

In the case of q=1, the position of substitution of —OR³, which is asubstituent of the benzothiazolyl group existing at the 2-position ofthe tetrazole skeleton, is not particularly limited, and the position ofsubstitution may be any one of the 4-position, 5-position, 6-position or7-position. From the viewpoint of the effect of shifting the maximumabsorption wavelength toward the longer wavelength side at the time ofchelation with a transition metal ion such as Ni²⁺, it is preferablethat the position of substitution of —OR³ is bonded to the 6-position ofthe benzothiazolyl group.

In the case of q=2, R³ represents a hydrogen atom, a methyl group, or anethyl group, and at least one is a methyl group or an ethyl group.Furthermore, when q is 2, these OR³'s may be disposed adjacently to eachother, and the OR³'s may be bonded to each other and form a ring. Inthis case, a suitable combination is a combination of a hydrogen atomand a methyl group for R³, or a combination of a methyl group and amethyl group for R³. In the case of q=2, the positions of substitutionof —OR³, which is a substituent of the benzothiazolyl group existing atthe 2-position of the tetrazole skeleton, are not particularly limitedas long as two —OR³'s are disposed adjacently to each other, and thepositions of substitution may be any one of the combinations of the4,5-positions, the 5,6-position, and the 6,7-position. From theviewpoint of the effect of shifting the maximum absorption wavelengthtoward the longer wavelength side at the time of chelation with atransition metal ion such as Ni²⁺, for the position of substitution ofat least one —OR³, it is preferable that —OR³ is bonded to the6-position of the benzothiazolyl group, that is, the positions ofsubstitution of two —OR³'s are preferably the 5,6-position, or the6,7-position. Specifically, in a case in which q is 2, it is preferablethat the substituted benzothiazolyl group existing at the 2-position ofthe tetrazole skeleton is any one of the following substituents.

Furthermore, when q is 2, in a case in which the substitutedbenzothiazolyl group existing at the 2-position of the tetrazoleskeleton is any one of the above-described substituents, it ispreferable that the substituted sulfonated phenyl group existing at the3-position of the tetrazole skeleton is a 4-methoxy-5-sulfophenyl group,from the viewpoint of the effect of shifting the maximum absorptionwavelength toward the longer wavelength side at the time of chelationwith a transition metal ion such as Ni²⁺.

In Formula (1), a substituted sulfonated phenyl group exists at the5-position of the tetrazole skeleton. R¹, which is a substituent for thesulfonated phenyl group, is any one selected from the group consistingof a hydrogen atom, a hydroxyl group, a methoxy group, and an ethoxygroup. From the viewpoint of enhancing the water-solubility of thetetrazolium salt and the formazan produced from the tetrazolium salt, R¹is preferably a hydrogen atom or a hydroxyl group. From the viewpointthat the compound can stably form a chelate with a transition metal ionin a wide pH region, R¹ is more preferably a hydrogen atom. Furthermore,the position of substitution in a case in which R¹ is a hydroxyl group,a methoxy group, or an ethoxy group is not particularly limited;however, the position of substitution is preferably the 4-position.

At the 5-position of the tetrazole skeleton, at least one sulfo group(—SO₃ ⁻) exists (m=1 or 2). It is considered that due to this sulfogroup, the water-solubility of the tetrazolium salt and the formazanproduced from the tetrazolium salt is enhanced. In regard to Formula(1), m represents the number of sulfo groups (—SO₃ ⁻) bonded to thephenyl group at the 5-position of the tetrazole skeleton, and is 1 or 2.Particularly, in a case in which a sulfo group is at the 2-position orthe 4-position, and in a case in which sulfo groups are at the2,4-position, a further enhancement of water-solubility can beexhibited. Furthermore, when sulfo groups are at the 2,4-position, it isadvantageous from the viewpoint that the synthesis of building blocksfor synthesizing the compound is easier. From the viewpoint that thecompound is highly water-soluble and can stably form a chelate compoundwith a transition metal ion in a wide pH region, or from the viewpointthat water-solubility is enhanced, it is preferable that m=2, and it ismore preferable that m=2, and R¹ represents a hydrogen atom.

At this time, in a case in which m=2, it is preferable that p, which isthe number of sites at which a sulfo group (—SO₃ ⁻) is bonded to thephenyl group at the 3-position of the tetrazole skeleton, is 1. Whensuch a number of substituents are selected, the water-solubility of thetetrazolium salt and the formazan produced therefrom is furtherenhanced.

In addition, from the viewpoint of water-solubility, it is preferable tosatisfy any one of the following items (1) to (4): (1) m=2, and p=1; (2)m=1, and n=0; (3) in regard to the phenyl group existing at the5-position of the tetrazole skeleton, R¹ is a hydroxyl group, and atthis time, a sulfo group (SO₃ ⁻) and a hydroxyl group are at the2,4-position or the 4- and 6-positions; and (4) p=0, and at least one ofR²'s is a carboxyl group. It is more preferable that (1) m=2, and p=1;or (4) p=0, and at least one of R²'s is a carboxyl group. In the item(3), it is speculated that since the sulfo group (SO₃ ⁻) and thehydroxyl group do not exist as adjacent substituents on the benzenering, there is no, or less, hydrogen bonding between the substituents,and the two substituents can efficiently contribute to water-solubility.

Here, the position of bonding of a sulfo group (—SO₃ ⁻) to the phenylgroup existing at the 5-position of the tetrazole skeleton is notparticularly limited. From the viewpoint of the effect of furtherenhancing the water-solubility of the tetrazolium salt and the formazanproduced from the tetrazolium salt, and from the viewpoint that themaximum absorption wavelength can be shifted toward the longerwavelength side, in the case of m=2, it is preferable that sulfo groups(—SO₃ ⁻) exist at the 2,4-position or the 3,5-position of the phenylgroup. It is particularly preferable that sulfo groups exist at the2,4-position of the phenyl group, and a tetrazolium salt compound thatdoes not precipitate even in the presence of a transition metal ion at ahigh concentration can be obtained. That is, by using this tetrazoliumsalt as an indicator reagent, a reagent composition for biologicalcomponent measurement that can be quantitatively determined even in acase in which the biological component is at a high concentration, canbe produced. That is, according to a preferred embodiment of the presentdisclosure, it is preferable that the phenyl group at the 5-position ofthe tetrazole skeleton is a phenyl group in which sulfo groups (—SO₃ ⁻)exist at the 2- and 4-positions.

In regard to Formula (1), a substituted phenyl group exists at the3-position of the tetrazole skeleton. Since the phenyl group isessentially substituted, n+p is 1 or greater.

R² as a substituent for the phenyl group at the 3-position of thetetrazole skeleton is any one selected from the group consisting of anitro group, —OR⁴, and a carboxyl group. From the viewpoint of theformability of a chelate between formazan and a transition metal ion, R²is preferably a nitro group or —OR⁴, and from the viewpoint ofwater-solubility, R² is preferably a carboxyl group. Furthermore, nrepresents the number of R² bonded to the phenyl group at the 3-positionof the tetrazole skeleton, and is an integer from 0 to 2. As describedbelow, by introducing R², the maximum absorption wavelength of thecompound can be moved to a longer wavelength range, or the stability ofthe compound can be enhanced. Therefore, it is preferable that n=1 or 2.In a case in which two R²'s exist, that is, n=2, R²'s may be identicalor different.

When n=1 or 2, it is preferable that at least one of R²'s is a —OR⁴group. That is, a suitable embodiment of the present disclosure is suchthat n is 1 or 2, and at least one of R²'s is a —OR⁴ group. When analkoxy group is introduced as a substituent of the phenyl group,stability of the compound is enhanced. From the viewpoint of enhancingthe water-solubility of the tetrazolium salt and the formazan producedfrom the tetrazolium salt, it is preferable that the —OR⁴ group is amethoxy group. Here, it is preferable that R⁴ is a methyl group or anethyl group, and from the viewpoint of water-solubility, it ispreferable R⁴ is a methyl group. In a case in which R⁴ is an alkyl grouphaving 3 or more carbon atoms, the tetrazolium salt and the formazanproduced from the tetrazolium salt have poor water-solubility, which isnot preferable.

The position of substitution of R² in a case in which n is 1 or 2 is notparticularly limited; however, it is preferable that the position ofsubstitution of the substituted sulfophenyl group existing at the3-position of the tetrazole skeleton is the 2-position, 3-position,4-position, 5-position, or 6-position; it is preferable that at leastone of R² is at the 2-position or the 4-position; and it is preferablethat the position of substitution is the 2-position and/or the4-position. By adopting such a structure, water-solubility is enhanced,and at the same time, the stability of the tetrazolium salt and theformazan produced from the tetrazolium salt can be enhanced.

p represents the number of sulfo groups (—SO₃ ⁻) bonded to the phenylgroup at the 3-position of the tetrazole skeleton, and is 0 or 1. Fromthe viewpoint of enhancing the water-solubility of the tetrazolium saltand the formazan produced from the tetrazolium salt, it is preferablethat p=1. In the case ofp=1, since a sulfo group is anelectron-withdrawing group, when there is another electron-withdrawinggroup (for example, a nitro group), the cationic charge of the nitrogenatom on the tetrazolium ring may be destabilized, and the stability ofthe compound may be deteriorated. As described above, stability of thecompound is enhanced by introducing an alkoxy group as a substituent ofthe phenyl group; however, when a nitro group is introduced at the sametime, an enhancement of the stability induced by the introduction of analkoxy group may not be exhibited. Therefore, from the viewpoint ofenhancing stability, in the case of p=1, n is 1 or 2, and preferably nis 1. Meanwhile, R² is preferably any one selected from the groupconsisting of —OR⁴ and a carboxyl group, and it is more preferable thatR² is —OR⁴. Alternatively, from the viewpoint of enhancing thewater-solubility of the tetrazolium salt and the formazan produced fromthe tetrazolium salt, it is preferable that p=0, and at least one ofR²'s is a carboxyl group. That is, according to a suitable embodiment,in regard to Formula (1), p is 1, or p=0 and at least one of R²'s is acarboxyl group. More suitably, m=2 and p=1, or p=0 and at least one ofR²'s is a carboxyl group.

In a case in which p=1, the position of substitution of the sulfo group(—SO₃ ⁻) that substitutes the phenyl group at the 3-position of thetetrazole skeleton is not particularly limited; however, it ispreferable that the position of substitution is the 3-position or the5-position. When the sulfo group is substituted at this position, thestability of the tetrazolium salt and the formazan produced from thetetrazolium salt can be enhanced more effectively.

Furthermore, in regard to Formula (1), the substituent existing at the3-position of the tetrazole skeleton is preferably a4-methoxy-3-sulfophenyl group, a 2-methoxy-5-sulfophenyl group, a2-methoxy-4-nitro-5-sulfophenyl group, a 2-methoxy-4-nitrophenyl group,a 4-sulfophenyl group, a 4-carboxy-2-methoxyphenyl group, a5-carboxy-2-methoxyphenyl group, a 3-carboxy-4-methoxyphenyl group, or a4-methoxy-5-sulfophenyl group; more preferably a 4-methoxy-3-sulfophenylgroup, a 2-methoxy-5-sulfophenyl group, a 3-carboxy-4-methoxyphenylgroup, or a 4-methoxy-5-sulfophenyl group; and particularly preferably a4-methoxy-3-sulfophenyl group, a 4-methoxy-5-sulfophenyl group, or a2-methoxy-5-sulfophenyl group. By adopting such structures, the colordevelopment sensitivity is increased, water-solubility is increased, andalso, the stability of the tetrazolium salt and the formazan producedfrom the tetrazolium salt can be enhanced. Furthermore, since themaximum absorption wavelength of the formazan compound itself can beshifted to a longer wavelength range, it is particularly preferable thatthe phenyl group existing at the 3-position of the tetrazole skeleton isa 4-methoxy-3-sulfophenyl group.

The total number of sulfo groups (m+p) existing in Formula (1) ispreferably 2 or greater, and more preferably 3, from the viewpoint ofenhancing the water-solubility of the tetrazolium salt and the formazanproduced from the tetrazolium salt.

In Formula (1) described above, X represents a hydrogen atom or analkali metal atom. Here, X exists in order to neutralize an anion (sulfogroup (—SO₃ ⁻)). Therefore, the type of the alkali metal is notparticularly limited and may be any one of lithium, sodium, potassium,rubidium, and cesium.

Preferred examples of the tetrazolium salt include the following.Meanwhile, in the structures described below, X represents an alkalimetal atom.

The method for producing the tetrazolium salt of the present disclosureis not particularly limited, and conventionally known methods can beapplied in a similar manner or after being appropriately modified. Forexample, hydrazone is synthesized by dehydration and condensation ofaldehyde and hydrazine, and then a corresponding diazonium salt iscaused to react in an aqueous solvent under basic conditions. Thereby,formazan is obtained. Here, for the basifying agent, sodium hydroxide,potassium hydroxide, and the like are used. Next, the formazan thusobtained is oxidized in an alcohol solvent (for example, methanol orethanol) using an oxidizing agent such as ethyl nitrite, butyl nitrite,or sodium hypochlorite, and a tetrazolium salt of Formula (1) can beobtained. According to an embodiment, a hydrazino-substitutedbenzothiazole having the following structure:

is caused to react with a substituted sulfonated benzaldehyde having thefollowing structure:

and a hydrazone compound is obtained having the following structure:

Meanwhile, while a substituted sulfonated aniline having the followingstructure:

is ice-cooled, hydrochloric acid is added thereto, and a sodium nitritesolution is added dropwise to the mixture. Thus, a benzenediazoniumchloride compound having the following structure:

is obtained. The hydrazone compound and the benzenediazonium chloridecompound obtained as described above are allowed to react under basicconditions (for example, in the presence of sodium hydroxide orpotassium hydroxide), and thereby a formazan compound having thefollowing structure:

is obtained. Next, the formazan compound obtained in this manner isoxidized in an alcohol solvent (for example, methanol or ethanol) usingan oxidizing agent (for example, a nitrous acid ester such as sodiumnitrite, ethyl nitrite, or butyl nitrite), and thereby the tetrazoliumsalt of the present disclosure is obtained.

The formazan produced from the tetrazolium salt of the presentdisclosure, or a chelate compound of formazan and a transition metal ionacquires the maximum absorption wavelength in a wavelength range (600 nmor greater, particularly 650 nm or greater) that does not overlap withthe main absorption band of hemoglobin, by being used alone or byforming a chelate compound with a transition metal compound.Furthermore, the tetrazolium salt of the present disclosure is highlywater-soluble. Therefore, the biological component concentration in abiological sample, particularly even in a whole blood sample, can bemeasured with high sensitivity by using the tetrazolium salt of thepresent disclosure. Specifically, the maximum absorption wavelength(λmax) of the formazan produced from the tetrazolium salt of the presentdisclosure or the chelate compound of formazan and a transition metalion is preferably 600 nm or greater, more preferably 630 nm or greater,and particularly preferably 650 nm or greater. When formazan or achelate compound of formazan and a transition metal ion (therefore, atetrazolium salt capable of producing such a formazan) having such amaximum absorption wavelength is used, the measurement is not likely tobe affected by the absorption of blood, and the biological componentconcentration can be measured more accurately with satisfactorysensitivity. Here, the upper limit of the maximum absorption wavelength(λmax) of the formazan produced from the tetrazolium salt of the presentdisclosure or the chelate compound of formazan and a transition metalion is not particularly limited; however, the upper limit is usually 900nm or less, and preferably 800 nm or less. Meanwhile, in the presentspecification, regarding the maximum absorption wavelength (λmax), avalue measured according to the method described in the followingEmbodiments will be employed.

Therefore, according to a second aspect of the present disclosure, thereis provided a reagent for biological component concentration measurementincluding 2-substituted benzothiazolyl-3-substitutedphenyl-5-substituted sulfonated phenyl-2H-tetrazolium salt of thepresent disclosure. Furthermore, according to a third aspect, there isprovided a method for measuring a biological component concentration,the method including adding 2-substituted benzothiazolyl-3-substitutedphenyl-5-substituted sulfonated phenyl-2H-tetrazolium salt of thepresent disclosure, an oxidoreductase, and a transition metal compoundto a biological sample, measuring the quantity of color development, andquantitatively determining the concentration of a biological componentin the biological sample based on the quantity of color development.

According to the present disclosure, the object of biological componentmeasurement is not particularly limited as long as the object includesthe intended biological component. Specific examples of the object ofbiological component measurement include blood as well as body fluidssuch as urine, saliva, and interstitial fluid. Furthermore, thebiological component is not particularly limited, and usually, abiological component that is measured by a colorimetric method or anelectrode method can be used similarly. Specific examples includeglucose, cholesterol, neutral lipids, nicotinamide adenine dinucleotidephosphate (NADPH), nicotinamide adenine dinucleotide (NADH), and uricacid. That is, according to a preferred embodiment of the second aspectof the disclosure, the reagent for biological component concentrationmeasurement of the present disclosure is used for the measurement of theconcentration of glucose, cholesterol, neutral lipids, nicotinamideadenine dinucleotide phosphate (NADPH), nicotinamide adeninedinucleotide (NADH), and uric acid in the blood or in a body fluid.Furthermore, according to a preferred embodiment of the third aspect ofthe present disclosure, the biological component in a biological sampleis glucose, cholesterol, neutral lipids, nicotinamide adeninedinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide(NADH), or uric acid in the blood or in a body fluid.

The reagent for biological component concentration measurement of thepresent disclosure essentially includes the tetrazolium salt of thepresent disclosure. As described above, in the reagent for biologicalcomponent concentration measurement, the formazan produced by areduction reaction of the tetrazolium salt has the maximum absorptionwavelength on the longer wavelength side, compared to the tetrazoliumsalts that are currently distributed. As the tetrazolium salt of thepresent disclosure produces a chelate compound with a transition metalion, the maximum absorption wavelength can be shifted toward the longerwavelength side. Therefore, particularly in the case of measuring thebiological component concentration in a whole blood sample, it ispreferable that the measurement system includes a transition metalcompound. That is, according to a preferred embodiment of the presentdisclosure, the reagent for biological component concentrationmeasurement further includes a transition metal compound. According tothis embodiment, even in the case of measuring the biological componentconcentration in a whole blood sample, formazan has the maximumabsorption wavelength in a wavelength range (600 nm or greater) thatdoes not overlap with the main absorption band of hemoglobin. Therefore,in a biological sample, particularly even in a whole blood sample, themeasurement sensitivity of the biological component concentration can befurther increased. The transition metal compound that can be used in acase in which the reagent for biological component concentrationmeasurement includes a transition metal compound is not particularlylimited. Specifically, a compound that can produce a transition metalion such as nickel ion (Ni²⁺), cobalt ion (Co²⁺), zinc ion (Zn²⁺), orcopper ion (Cu²⁺), can be used. When such an ion is used, the maximumabsorption wavelength of formazan can be further shifted toward thelonger wavelength side. Among these, nickel ion is preferred. Sincenickel ion is not likely to be subjected to the action of oxidation orreduction, the error of measurement can be reduced more effectively.That is, according to a preferred embodiment of the present disclosure,the transition metal compound is a nickel compound. Furthermore, thecompound that produces the transition metal ion is not particularlylimited; however, a compound that produces ions in an aqueous liquid(for example, water, a buffer solution, blood, or a body fluid) ispreferred. Examples thereof include chlorides, bromides, sulfates, andorganic acid salts of the above-mentioned transition metals. Thetransition metal compounds described above may be used singly or incombination of two or more kinds thereof. Furthermore, according to thepresent embodiment, the content of the transition metal compound is notparticularly limited; however, the content can be appropriately selectedaccording to the desired maximum absorption wavelength of the formazancompound. Specifically, the content of the transition metal compound ispreferably an amount such that the proportion of the transition metal(transition metal ions) is 0.1 to 10 mol, and more preferably 0.5 to 4mol, with respect to 1 mol of the tetrazolium salt. With such an amount,the maximum absorption wavelength of the formazan compound can beshifted to a desired wavelength range.

The reagent for biological component concentration measurement mayfurther include other components, in addition to the transition metalcompound or instead of the transition metal compound. Here, regardingthe other components, usually, components that are appropriatelyselected according to the type of the biological component as an objectof measurement and are added for the purpose of measuring theconcentration of the biological component, can be similarly used.Specific examples include an oxidoreductase, an electron carrier, a pHbuffering agent, and a surfactant. Here, the above-mentioned othercomponents may be respectively used singly, or two or more kinds thereofmay be used in combination. Furthermore, each of the above-mentionedother components may be used singly or in combination of two or morekinds thereof.

Here, the oxidoreductase is not particularly limited and can beappropriately selected depending on the type of the biological componentas an object of measurement. Specific examples include glucosedehydrogenases such as a glucose dehydrogenase (GDH), a glucosedehydrogenase that uses pyrroloquinoline quinone (PQQ) as a coenzyme(PQQ-GDH), a glucose dehydrogenase that uses flavin adenine dinucleotide(FAD) as a coenzyme (GDH-FAD), a glucose dehydrogenase that usesnicotinamide adenine dinucleotide (NAD) as a coenzyme (GDH-NAD), and aglucose dehydrogenase (GDH) that uses nicotine adenine dinucleotidephosphate (NADP) as a coenzyme (GDH-NADP) or the like; glucose oxidase(GOD), lactic acid dehydrogenase (LDH), cholesterol dehydrogenase,cholesterol oxidase, and uric acid dehydrogenase. Here, theoxidoreductases may be used singly, or two or more kinds thereof may beused in combination. For example, in a case in which the biologicalcomponent is glucose, it is preferable that the oxidoreductase is aglucose dehydrogenase or a glucose oxidase. In a case in which thebiological component is cholesterol, it is preferable that theoxidoreductase is a cholesterol dehydrogenase or a cholesterol oxidase.The content of the oxidoreductase in a case in which the reagent forbiological component concentration measurement includes anoxidoreductase is not particularly limited, and the content can beselected as appropriate according to the amount of the tetrazolium salt.The electron carrier is not particularly limited, and any known electroncarrier may be used. Specific examples include a diaphorase, phenazinemethosulfate (PMS), 1-methoxy-5-methylphenazinium methyl sulfate(1-Methoxy PMS or m-PMS), nicotinamide adenine dinucleotide phosphate(NADPH), and nicotinamide adenine dinucleotide (NADH). Here, theelectron carriers may be used singly or in combination of two or morekinds thereof. The content of the electron carrier in a case in whichthe reagent for biological component concentration measurement includesan electron carrier is not particularly limited, and the content can beselected as appropriate according to the amount of the tetrazolium salt.For example, the content of the electron carrier is preferably 0.05% to10% by mass, and more preferably 0.1% to 5% by mass, with respect to thetetrazolium salt. With such an amount, the reduction reaction can becarried out more efficiently.

In regard to the description given above, it is preferable that thereagent for biological component concentration measurement furtherincludes a transition metal compound and an oxidoreductase, and it ismore preferable that the reagent further includes a transition metalcompound, an oxidoreductase, and an electron carrier. That is, accordingto a preferred embodiment of the present disclosure, the reagent forbiological component concentration measurement includes the2-substituted benzothiazolyl-3-substituted phenyl-5-substitutedsulfonated phenyl-2H-tetrazolium salt of the present disclosure, atransition metal compound, and an oxidoreductase. Furthermore, accordingto a more preferred embodiment of the present disclosure, the reagentfor biological component concentration measurement includes the2-substituted benzothiazolyl-3-substituted phenyl-5-substitutedsulfonated phenyl-2H-tetrazolium salt of the present disclosure, atransition metal compound, an oxidoreductase, and an electron carrier.Here, in a case in which the biological component is β-D-glucose, thetransition metal compound is nickel chloride, the oxidoreductase is aglucose dehydrogenase that uses flavin adenine dinucleotide as acoenzyme (GDH-FAD), and the electron carrier is1-methoxy-5-methylphenazium methyl sulfate (m-PMS), first, β-D-glucoseand m-PMS are subjected to the action of GDH-FAD to become gluconic acidand reduced m-PMS, respectively, and this reduced m-PMS and thetetrazolium salt produce m-PMS and formazan. Thus, color developmentoccurs. Furthermore, this formazan forms a chelate compound with nickelion, and thereby the maximum absorption wavelength is shifted toward thelonger wavelength range side (for example, 600 nm or greater). For thisreason, when the reagent for biological component concentrationmeasurement of the present disclosure is used, the influence exerted bythe absorption of hemoglobin can be reduced, and therefore, thebiological component concentration in a biological sample, particularlyeven in a whole blood sample, can be measured with high sensitivity.

The form of use of the reagent for biological component concentrationmeasurement is not particularly limited and may be in any one of a solidform, a gel form, a sol form, and a liquid form. The reagent forbiological component concentration measurement may also include water, abuffer, a surfactant, and the like. Here, the buffer is not particularlylimited, and those buffers generally used at the time of measuring abiological component concentration can be similarly used. Specificexamples that can be used include “GOOD” buffers such as a phosphatebuffer, a citrate buffer, a citrate-phosphate buffer, atris(hydroxymethyl)aminomethane HCl buffer (Tris hydrochloride buffer),an MES buffer (2-morpholinoethane sulfonate buffer), a TES buffer(N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate buffer), an acetatebuffer, a MOPS buffer (3-morpholinopropane sulfonate buffer), aMOPS-NaOH buffer, a HEPES buffer (4-(2-hydroxyethyl)-1-piperazinethanesulfonate buffer), and a HEPES-NaOH buffer; amino acid-based bufferssuch as a glycine-hydrochloride buffer, a glycine-NaOH buffer, aglycylglycine-NaOH buffer, and a glycylglycine-KOH buffer; boricacid-based buffers such as a Tris borate buffer, a borate-NaOH buffer,and a borate buffer; and an imidazole buffer. Among these, a phosphatebuffer, a citrate buffer, a citrate-phosphate buffer, a Trishydrochloride buffer, a MES buffer, a citrate buffer, a MOPS buffer, anda HEPES-NaOH buffer are preferred. Here, the concentration of the bufferis not particularly limited; however, it is preferable that theconcentration is 0.01 to 1.0 M. Meanwhile, the concentration of thebuffer according to the present disclosure refers to the concentrationof a buffer included in an aqueous solution (M, mol/L). Furthermore, itis preferable that the pH of the buffer solution does not have anyaction on the biological component. From the above-described viewpoint,it is preferable that the pH of the buffer solution is near neutrality,for example, about 5.0 to 8.0. In a case in which the reagent forbiological component concentration measurement is liquid, theconcentration of the tetrazolium salt is not particularly limited aslong as it is a concentration at which the concentration of a desiredbiological component can be measured; however, it is preferable that thetetrazolium salt is included in a sufficient amount with respect to theabundance of the desired biological component. When the above-describedviewpoints and the conventional biological component concentrations tobe measured are taken into consideration, the concentration of thetetrazolium salt is preferably 0.01 to 0.2 mol/L, and more preferably0.05 to 0.1 mol/L, with respect to the reagent for biological componentconcentration measurement. With such an amount, the tetrazolium saltreacts with substantially the entire amount (for example, 95 mol % ormore, preferably 98 mo % or more, and particularly preferably 100 mol %)of the biological component included in a biological sample. Therefore,the desired biological component concentration can be measuredaccurately and rapidly with satisfactory sensitivity.

By using the reagent for biological component concentration measurementof the present disclosure, the concentration of a particular biologicalcomponent included in a biological sample can be measured withsatisfactory sensitivity. Here, the measurement method is notparticularly limited and can be selected as appropriate according to thetype of the biological component as an object of measurement. Forexample, in a case in which the biological component is β-D-glucose, andthe oxidoreductase is a glucose dehydrogenase (GDH), glucose is oxidizedby GDH, thereby gluconic acid is produced, and reduction of a coenzymeof GDH or an electron carrier substance at that time is utilized. Thus,specifically, the measurement methods are roughly classified intomethods of photometrically measuring the degree of coloring of theconsequently reduced tetrazolium salt (therefore, formazan or a chelatecompound of formazan and a transition metal ion) (colorimetric methods),and methods of measuring the electric current produced by anoxidation-reduction reaction (electrode methods). Among the methodsdescribed above, the measurement of the blood sugar level according to acolorimetric method has advantages such as easy implementation ofcorrection using the hematocrit value at the time of calculating theblood sugar level, and simple and easy production process. Therefore,the reagent for biological component concentration measurement can besuitably used for a colorimetric method. Particularly, in the case ofmeasuring the glucose concentration in a whole blood sample, acolorimetric method is preferably used.

The reagent for biological component concentration measurement of thepresent disclosure may be used in the form as received for themeasurement of a biological component concentration; however, thereagent may also be incorporated into a chip for biological componentconcentration measurement. That is, the present disclosure also providesa chip for biological component concentration measurement including thereagent for biological component concentration measurement of thepresent disclosure (hereinafter, also simply referred to as “sensorchip”). The reagent or method for biological component concentration ofthe present disclosure can be incorporated into an automatic analyzer,an analytic kit, a simplified blood glucose meter, or the like and usedfor common clinical examinations. Furthermore, it is also possible toincorporate the reagent of the present disclosure into a commerciallyavailable biosensor. In the case of incorporating the reagent forbiological component concentration measurement into a chip forbiological component concentration measurement, the content of thereagent per chip is not particularly limited, and an amount that isconventionally used in the pertinent art can be similarly employed.However, it is preferable that the tetrazolium salt is included in asufficient amount with respect to the abundance of a desired biologicalcomponent. When the above-described viewpoints and the conventionalbiological component concentrations to be measured are taken intoconsideration, the concentration of the tetrazolium salt is preferably 3to 50 nmol, and more preferably 10 to 30 nmol, per chip. With such anamount, the tetrazolium salt reacts according to the amounts ofsubstantially all the biological components included in a biologicalsample. Therefore, a desired biological component concentration can bemeasured accurately and rapidly with satisfactory sensitivity.

In the following description, the form of the sensor chips of thepresent disclosure used for measuring the blood sugar level by acolorimetric method (colorimetric blood glucose meter) will be explainedwith reference to the drawings. However, the present disclosure ischaracterized by the use of the reagent for biological componentconcentration measurement of the present disclosure, and the structureof the chip is not particularly limited. Therefore, the reagent forbiological component concentration measurement of the present disclosuremay also be applied to a commercially available sensor chip or the chipsdescribed in WO 2014/04970 A, WO 2016/051930 A, and the like. Similarly,in the embodiment described below, a specific form of a chip intendedfor the measurement of the blood sugar level will be explained; however,the sensor chip is not limited to the relevant use and can be applied inthe same manner, or after being appropriately modified, to other useapplications.

FIG. 9 is a plan view schematically illustrating a blood glucose meterused for the detection of glucose (blood sugar) using the sensor chipaccording to the present embodiment.

In FIG. 9, the blood glucose meter 10 is configured as an instrument formeasuring glucose (blood sugar) in a blood sample. This blood glucosemeter 10 can be used mainly for personal use, in which the instrument isoperated by the user (testee). The user can implement the blood sugarmanagement of oneself by measuring the preprandial blood sugar.Furthermore, a health care worker may use the blood glucose meter 10 inorder to evaluate the health status of a testee, and in this case, theblood glucose meter 10 may be configured to be installable in a medicalfacility or the like by being modified as appropriate.

The blood glucose meter 10 employs the principle of a colorimetricmethod, by which the content of glucose in a blood sample (blood sugarlevel) is photometrically measured. Particularly, this blood glucosemeter 10 performs the measurement of blood sugar by means of atransmission type measuring unit 14 that irradiates an analysis sample(blood) with measuring light having a predetermined wavelength andreceiving the light transmitted through the analysis sample.

In the blood glucose meter 10, a sensor chip 12 having bloodincorporated therein is mounted, or blood is incorporated into thesensor chip 12 in a state of having the sensor chip 12 mounted, andthereby glucose is detected using the measuring unit 14. The sensor chip12 may be configured into a disposable type that is disposed of afterevery single measurement. Meanwhile, it is preferable that the bloodglucose meter 10 is configured into a portable and tenacious instrumentso that the user can repeat measurement simply and easily.

The sensor chip 12 includes, as illustrated in FIG. 10, a chip main body18 formed into a plate shape, and a cavity 20 (cavity for liquid)extending in the planar direction of the plate surface in the interiorof the chip main body 18.

As illustrated in FIG. 10, the chip main body 18 is formed into arectangular shape having long edges 22 that are elongated in thedirection of plugging and unplugging of the blood glucose meter 10(direction of the front end and the base end of the blood glucose meter10, that is, direction B) and also having short edges 24 that are shortin direction A. For example, it is desirable that the length of the longedges 22 of the chip main body 18 is set to a length that is two or moretimes the length of the short edges 24. Thereby, in the sensor chip 12,a sufficient amount of insertion is secured for the blood glucose meter10.

Furthermore, the thickness of the chip main body 18 is formed to be verysmall (thin) compared to the lateral surfaces formed into a rectangularshape (in FIG. 10, it is deliberately depicted such that the chip mainbody has a sufficient thickness). For example, the thickness of the chipmain body 18 is preferably set to be 1/10 or less of the length of theshort edges 24. The thickness of this chip main body 18 may be designedas appropriate according to the shape of the insertion port 58 of theblood glucose meter 10.

In the sensor chip 12, the chip main body 18 is configured to include apair of plate pieces 30 and a pair of spacers 32 so that the sensor chip12 has a cavity 20.

FIG. 11 is a top view diagram illustrating the sensor chip of FIG. 9. InFIG. 11, the corners of the chip main body 18 are sharp; however, forexample, the corners may be formed into round corners. Furthermore, thechip main body 18 is not limited to a thin plate shape, and the shapemay be definitely designed freely. For example, the chip main body 18may be formed into a square shape, another polygonal shape, a circularshape (including an elliptical shape), or the like as viewed from thetop.

The cavity 20 provided inside the chip main body 18 is positioned in themiddle in the minor axis direction of the chip main body 18, and isformed into a linear shape along the longitudinal direction of the chipmain body 18. This cavity 20 extends into the front end port 20 a formedat the front end edge 24 a of the chip main body 18 and the base endport 20 b formed at the base end edge 24 b, respectively, and the cavityis in communication with the outside of the chip main body 18. In thecavity 20, when the user's blood is introduced thereinto through thefront end port 20 a, the blood can be caused to flow along the directionof extension based on the capillary phenomenon. The quantity of theblood that flows through the cavity 20 is small, and even if the bloodmoves to the base end port 20 b, leakage is suppressed by tension. Onthe side of the base end edge 24 b of the chip main body 18, anabsorptive portion that absorbs blood (for example, the spacers 32 thatwill be described below is formed from a porous material only on thebase end side) may be provided.

Furthermore, at a predetermined position of the cavity 20 (for example,a position slightly shifted to the base end from the midpoint betweenthe front end port 20 a and the base end port 20 b illustrated in FIG.11), a measuring object portion 28 in which a reagent (indicatorreagent) 26 that develops a color correspondingly to the glucose (bloodsugar) concentration in the blood by reacting with glucose in the blood(blood sugar) is applied, and measurement is achieved by the bloodglucose meter 10, is provided. The blood flowing through the cavity 20in the direction of the base end comes into contact with the reagent 26applied on the measuring object portion 28, and the measuring objectportion 28 is colored as the blood reacts with the reagent 26.Meanwhile, in the longitudinal direction of the cavity 20, the positionof application of the reagent 26 and the measuring object portion 28 maybe shifted from each other, and for example, a reaction part appliedwith the reagent 26 may be provided on the upstream side in the bloodflow direction of the measuring object portion 28.

In the sensor chip 12, the chip main body 18 is composed of a pair ofplate pieces 30 and a pair of spacers 32 so as to have the cavity 20described above. The plate pieces 30 that form a pair are respectivelyformed into the rectangular shape described above as viewed from thelateral side and are disposed in a mutually laminating direction. Thatis, a pair of plate pieces 30 constitutes the two lateral surfaces(upper surface and lower surface) of the chip main body 18. The platethickness of each plate piece 30 is very small, and for example, theplate thickness may be set to have the same dimension of about 5 to 50μm. The thickness of two (a pair) of the plate pieces 30 may bedifferent from each other.

The pair of plate pieces 30 has a strength that maintains the plateshape and does not undergo plastic deformation even if a certain degreeof pressing force is applied in a direction orthogonally intersectingthe planar direction. Furthermore, each of the plate pieces 30 includesa transparent part or a translucent portion so that the measuring lightcan be transmitted. Furthermore, it is preferable that each of the platepieces 30 is formed on a flat plate surface having appropriatehydrophilicity so that blood can be caused to flow in the cavity 20.

The material that constitutes the plate pieces 30 is not particularlylimited; however, a thermoplastic resin material, glass, quartz, or thelike may be applied. Examples of the thermoplastic resin materialinclude polymeric materials such as a polyolefin (for example,polyethylene or polypropylene), a cycloolefin polymer, a polyester (forexample, polyethylene terephthalate or polyethylene naphthalate),polyvinyl chloride, polystyrene, an ABS resin, an acrylic resin, apolyamide, and a fluororesin; and mixtures thereof.

Furthermore, a pair of spacers 32 is disposed so as to be interposedbetween the pair of plate pieces 30, and the spacers are stronglyadhered to the respective facing surfaces of the plate pieces 30 bymeans of a predetermined joining means (adhesive or the like). That is,the spacers 32 are members that are disposed between the plate pieces 30constituting a pair so as to separate apart the plate pieces, andthereby form the cavity 20 between the pair of plate pieces 30 and thepair of spacers 32 themselves. In this case, one of the spacers 32 isdisposed so as to come into contact with the upper long edge 22 a of thechip main body 18 in FIG. 11 and to extend in the direction from thefront end to the base end along this upper long edge 22 a. The otherspacer 32 is disposed so as to come into contact with the lower longedge 22 b of the chip main body 18 in FIG. 11 and to extend in thedirection from the front end to the base end along this lower long edge22 b.

The material (base material) that constitutes a pair of spacers 32 isnot particularly limited; however, examples include various elastomerssuch as a styrene-based elastomer, a polyolefin-based elastomer, apolyurethane-based elastomer, a polyester-based elastomer, apolyamide-based elastomer, a polybutadiene-based elastomer, atrans-polyisoprene-based elastomer, a fluorine rubber-based elastomer,and a chlorinated polyethylene-based elastomer. Alternatively, inaddition to the thermoplastic elastomers, various materials capable ofelastic deformation may be applied, or structures such as a porous body(for example, sponge) capable of elastic deformation may also beapplied. Furthermore, the spacers may also be applied as spacers 32having, on one surface or both surfaces of the base material, anadhesive that adheres the plate pieces 30 by being brought into a curedstate or a semicured state between the pair of plate pieces 30.Furthermore, the spacers 32 may also be configured such that the spacerscontain the reagent 26 and elute the reagent 26 into the cavity 20.

The plate pieces 30 or the spacers 32 may be hydrophilization-treatedmaterials. Examples of the method of performing hydrophilizationtreatment include a method of applying an aqueous solution containing ahydrophilic polymer such as a surfactant, polyethylene glycol,polypropylene glycol, hydroxypropyl cellulose, a water-soluble silicone,polyacrylic acid, polyvinylpyrrolidone, or polyacrylamide by animmersion method, a spraying method, or the like; and methods of plasmairradiation, glow discharge, corona discharge, ultraviolet irradiation(for example, excimer light irradiation), and the like. These methodsmay be used singly or in combination.

Next, the apparatus main body 16 of the blood glucose meter 10 will beexplained. As illustrated in FIG. 9, the blood glucose meter 10 has acase 40 that constitutes the external appearance. The case 40 includes abox body 44 that accommodates a control unit 42 of the blood glucosemeter 10 inside the box body into a size that can be easily gripped andoperated by the user; and a cylindrical-shaped photometric unit 46 thatprotrudes from one edge (front end side) of the box body 44 to the frontend direction and accommodates a photometric measuring unit 14 in theinside. Furthermore, on the upper surface of the box body 44, a powersupply button 48, an operation button 50, and a display 52 are provided,and on the upper surface of the photometric unit 46, an ejection lever54 is provided.

The power supply button 48 switches the operation start and stop of theblood glucose meter 10 through the operation of the user. In regard tothe blood glucose meter 10 in an activating state, the operation button50 functions as an operation unit that performs measurement or displayof the blood sugar level based on the operation of the user, switchesthe display of the measurement results (including the past measurementresults), and the like. The display 52 is configured to include liquidcrystals, organic EL, or the like, and displays the information suppliedto the user in a measurement operation, such as the display ofmeasurement results or the display of errors.

The ejection lever 54 is provided so as to be movable in the directionfrom the front end to the base end, and ejection level releases the lockof an ejection pin that is not shown in the diagram and is providedinside the photometric unit 46, and thereby enables the ejection pin tomove in the direction toward the front end.

On the other hand, the photometric unit 46 of the apparatus main body 16is extended long from the box body 44 in the direction toward the frontend, in order to press the front end with the user's finger or the like.As illustrated in FIG. 10, a chip mounting part 60 having an insertionport 58; and a measuring unit 14 that photometrically detects glucose(blood sugar) in the blood are provided in this photometric unit 46.

The chip mounting part 60 is formed from a material having high hardness(rigidity) (for example, stainless steel) into a cylindrical shape thatincludes a flange portion 60 a protruding outward on the front end sideand has a predetermined length in the axial direction. This chipmounting part 60 is positioned and fixed over the front end surface andthe axial center (central portion) of the photometric unit 46 formedfrom a resin material. On the inner surface of the photometric unit 46,a fixing wall 46 a that strongly fixes the chip mounting part 60 isformed in a protruding manner, as illustrated in FIG. 12A.

Regarding the material that constitutes the chip mounting part 60, forexample, a material that is hard, does not easily undergo dimensionalchange, is not likely to be abraded even if plugging and unplugging ofthe sensor chip is repeatedly performed, and can be processed withsatisfactory dimensional accuracy, such as a metal such as stainlesssteel or titanium; alumite coating-treated aluminum; a liquid crystalpolymer; a plastic having a filler such as glass or mica incorporatedtherein; a plastic having its surface covered with a cured coating filmby nickel plating or the like; carbon fibers; or fine ceramics, may bementioned. Among these, when a metal material is applied, an insertionport 58 can be molded easily with high dimensional accuracy at the timeof producing the chip mounting part 60 (injection molding, pressmolding, or the like). Meanwhile, the apparatus main body 16 may beintegrally molded with the chip mounting part 60 by configuring thephotometric unit 46 itself with a hard material (for example, a metalmaterial).

At the axial center of the chip mounting part 60, an insertion port 58is provided as the axis center is surrounded by walls 62 of this chipmounting part 60. The insertion port 58 is formed into a rectangularshape having a cross-section that is long in the direction of insertion(direction B) and is short in the lateral width direction (direction A).The insertion port 58 has a predetermined depth from the front endsurface toward the inner side (direction to the base end) in a state inwhich the chip mounting part 60 is fixed to the photometric unit 46.

On the front end side of the chip mounting part 60, an insertion opening58 a that is connected to the insertion port 58 and is also incommunication with the outside, is formed. The dimension in thedirection of insertion (direction B) of this insertion opening 58 acoincides with the dimension of the short edge 24 of the sensor chip 12(length in direction A). Furthermore, the dimension in the lateral widthdirection of the insertion opening 58 a, that is, the distance between apair of walls 62 constituting the side surfaces of the insertion port58, is substantially the same as the thickness in the direction oflamination of the sensor chip 12 (Tall in FIG. 12A), as illustrated inFIG. 12A.

The chip mounting part 60 forms, together with the fixing wall 46 a ofthe photometric unit 46, a pair of device accommodating space 64 at aposition in the middle of the extension of the insertion port 58 (portfor measurement 59). The pair of device accommodating spaces 64 is aportion of the measuring unit 14, and the spaces are provided atpositions facing each other, with the insertion port 58 disposedtherebetween. The device accommodating spaces are each in communicationwith the port for measurement 59 through a light guide 66 formed by thechip mounting part 60.

The measuring unit 14 constitutes a light emitting unit 70 byaccommodating a light emitting device 68 in one of the deviceaccommodating spaces 64, and constitutes a light receiving unit 74 byaccommodating a light receiving device 72 in the other deviceaccommodating space 64. The light guides 66 of the chip mounting part 60plays the role of a so-called aperture by being formed into acircular-shaped hole having an appropriate diameter.

The light emitting device 68 of the light emitting unit 70 includes afirst light emitting device 68 a that irradiates the sensor chip 12 withmeasuring light having a first wavelength; and a second light emittingdevice 68 b that irradiates the sensor chip 12 with measuring lighthaving a second wavelength different from the first wavelength (notillustrated in FIG. 10). The first light emitting device 68 a and thesecond light emitting device 68 b are provided in parallel at thepositions facing the light guides 66 of the device accommodating spaces64.

The light emitting device 68 (first and second light emitting devices 68a and 68 b) can be constructed from light emitting diodes (LED). Thefirst wavelength is a wavelength for detecting the coloration density ofthe reagent 26 in accordance with the amount of blood sugar, and forexample, the first wavelength is 600 nm to 680 nm. The second wavelengthis a wavelength for detecting the red blood cell concentration in theblood, and for example, the second wavelength is 510 nm to 540 nm. Thecontrol unit 42 inside the box body 44 supplies a driving current andthereby causes the first and second light emitting devices 68 a and 68 bto emit light respectively at predetermined timings. In this case, theblood sugar level obtainable from the coloration density is correctedusing the hematocrit value obtainable from the red blood cellconcentration, and the blood sugar level is determined. Meanwhile, it isalso acceptable to compensate for noises attributed to blood cells byfurther making measurements at different measurement wavelengths.

The light receiving unit 74 is configured by disposing one lightreceiving device 72 at a position facing the light guide 66 of thedevice accommodating space 64. This light receiving unit 74 is toreceive the transmitted light coming from the sensor chip 12, and forexample, the light receiving unit 74 can be composed of a photodiode(PD).

Furthermore, the at the bottom (base end surface) of the insertion port58, the ejection pin 56 (ejection part) connected to the ejection lever54 is provided. The ejection pin 56 includes a rod portion 56 aextending along the axial direction of the photometric unit 46; and areceptor 56 b having a large diameter on the outer side in thecircumferential direction at the tip of the rod portion 56 a. Thereceptor 56 b is brought into contact with the base end edge 24 b of thesensor chip 12 inserted into the insertion port 58. Furthermore, betweenthe bottom of the insertion port 58 and the receptor 56 b of theejection pin 56, a coil spring 76 surrounding the ejection pin 56 in anon-contacting manner is provided. The coil spring 76 elasticallysupports the receptor 56 b of the ejection pin 56.

When insertion of the sensor chip 12 is completed, as illustrated inFIG. 12B, the measuring object portion 28 of the sensor chip 12 isdisposed at the position that overlaps with the light guides 66.

The ejection pin 56 is displaced in the direction toward the base end asthe receptor 56 b is pushed along with the insertion of the sensor chip12 by the user, and the ejection pin 56 is locked (fixed) by a lockingmechanism that is provided inside the case 40 and is not shown in thediagram. The coil spring 76 elastically contracts along with thedisplacement of the receptor 56 b. Then, when the ejection pin 56slightly moves as a result of the user's operation of the ejection lever54, the lock of the locking mechanism is released, and the sensor chipslides in the direction toward the front end by the elastic restoringforce of the coil spring 76. Thereby, the sensor chip 12 is pushed outto the ejection pin 56 and is taken out through the insertion port 58.

Returning to FIG. 9, the control unit 42 of the apparatus main body 16is composed of a control circuit having, for example, a computationunit, a memory unit, and an input/output unit. For this control unit 42,a well-known computer can be applied. The control unit 42 derives andcontrols the measuring unit 14 under, for example, the user's operationof the operation button 50, detects and calculates the glucose level inblood, and displays the blood sugar level thus calculated, on thedisplay 52.

For example, in regard to a blood glucose meter 10 that transmitsmeasuring light through a sensor chip 12 and thereby measures an objectof analysis (for example, glucose), the control unit 42 calculates themeasurement results based on the Beer-Lambert law expressed by thefollowing Formula (A).

[Math. 1]

log₁₀(l ₁ /l ₀)=−αL  (A)

In Formula (A) shown above, l₀ represents the intensity of light beforeentering a blood sample; l₁ represents the intensity of light afterbeing emitted from the blood sample; α represents the extinctioncoefficient; and L represents the distance (cell length) through whichthe measuring light passes.

EXAMPLES

The effects of the present disclosure will be described using thefollowing Examples and Comparative Examples. However, the technicalscope of the present disclosure is not intended to be limited only tothe following Examples. In the Examples described below, unlessparticularly stated otherwise, the operation was carried out at roomtemperature (25° C.). Furthermore, unless particularly stated otherwise,the units “percent (o)” and “parts” mean “percent by mass (mass %)” and“parts by mass”, respectively.

Example 1: Synthesis of Tetrazolium Compound 1

A compound (tetrazolium compound 1) having the following structure wassynthesized according to the following method.

1. Synthesis of Hydrazone Compound 1

1.59 g of disodium 4-formylbenzene-1,3-disulfonate (manufactured byTokyo Chemical Industry Co., Ltd.) and 1.0 g of(6-methoxy-1,3-benzothiazol-2-yl) hydrazine (also known as2-hydrazino-6-methoxy-1,3-benzothiazole) (manufactured by Santa CruzBiotechnology, Inc.) were suspended in 60 mL of RO water. Thissuspension was heated and stirred in a water bath at 60° C. for 2 hoursunder the acidity of acetic acid. After completion of the heating andstirring, the solvent was removed. This residue was washed withisopropanol, and then a precipitate was separated by filtration. Thisprecipitate was dried in a draught, and thereby hydrazone compound 1 wasobtained. 1.8 g of the compound was recovered, and the yield was 70% bymass.

2. Synthesis of Formazan Compound 1

0.76 g of the hydrazone compound 1 of the above section 1. was dissolvedin a mixed liquid of 10 mL of RO water and 10 mL of DMF, and thereby asolution of hydrazone compound 1 was produced. 0.264 g ofp-anisidine-3-sulfonic acid (manufactured by Tokyo Chemical IndustryCo., Ltd.) was suspended in 4.09 mL of RO water, and 130 μL of 10 N NaOHwas added to the suspension to dissolve therein. While this solution asmaintained at 0° C., 280 μL of 9.6 N HCl was added thereto, a sodiumnitrite solution was added dropwise thereto, and thus diazotization wasperformed. This diazotized solution was maintained at −20° C., and thissolution was added dropwise to the hydrazone compound 1 solution. Aftercompletion of the dropwise addition, 300 μL of 10 N NaOH was addeddropwise thereto, the mixture was stirred at room temperature (25° C.)for 2 hours, and thus a solution including formazan compound 1 (formazancompound 1 solution) was produced. The pH of this formazan compound 1solution was adjusted to neutrality with 9.6 N HCl, and the solvent wasremoved. The residue thus obtained was washed with isopropanol, and thena precipitate was separated by filtration. This precipitate was dried,and formazan compound 1 was obtained.

3. Purification of Formazan Compound 1 and Synthesis of TetrazoliumCompound 1

The formazan compound 1 of the above section 2. was dissolved in 10 mLof RO water, and thereby a formazan compound 1 solution was produced. Adisposable column (size: 20 cm×5 cm) was packed with a filler for columnchromatography (manufactured by NACALAI TESQUE, INC., COSMOSIL40C₁₈-PREP), and the disposable column was mounted in a columnpreparative separation system (manufactured by BÜCHI Labortechnik AG,trade name: SEPACORE). The formazan compound 1 solution was purifiedusing this column system. The solvent of a fraction thus collected wasremoved, and to the solid component thus obtained, 15 mL of methanol,250 μL of 9.6 N HCl, and 5 mL of a 15% ethyl nitrite (CH₃CH₂NO₂)-ethanolsolution were added. The mixture was stirred for 72 hours at roomtemperature (25° C.) in the dark.

4. Collection of Tetrazolium Compound 1

Diethyl ether was added to the reaction solution of the above section3., and thereby tetrazolium compound 1 was precipitated. Thisprecipitate was centrifuged, the supernatant was removed, and then theresidue was further washed with diethyl ether. The precipitate thusobtained was dried in a draught, and thus tetrazolium compound 1 wasobtained (120 mg, yield: 11.8% by mass).

Example 2: Synthesis of Tetrazolium Compound 2

A compound having the following structure (tetrazolium compound 2) wassynthesized according to the following method.

1. Synthesis of Hydrazone Compound 1

Hydrazone compound 1 was synthesized in the same manner as in 1.Synthesis of hydrazone compound 1 of Example 1.

2. Synthesis of Formazan Compound 2

0.76 g of the hydrazone compound 1 was dissolved in a mixed liquid of 10mL of RO water and 10 mL of DMF, and thereby a hydrazone compound 1solution was produced. 0.264 g of o-anisidine-5-sulfonic acid(manufactured by Tokyo Chemical Industry Co., Ltd.) was suspended in4.09 mL of RO water, and 130 μL of 10 N NaOH was added to the suspensionto dissolve therein. While this solution was maintained at 0° C., 280 μLof 9.6 N HCl was added to the solution, a sodium nitrite solution wasadded dropwise thereto, and diazotization was performed. This diazotizedsolution was maintained at −20° C., and this solution was added dropwiseto the hydrazone compound 1 solution. After completion of the dropwiseaddition, 300 μL of 10 N NaOH was added dropwise thereto, the mixturewas stirred at room temperature (25° C.) for 2 hours, and thereby asolution including formazan compound 2 (formazan compound 2 solution)was produced. The pH of this formazan compound 2 solution was adjustedto neutrality with 9.6 N HCl, and the solvent was removed. The residuethus obtained was washed with isopropanol, and then a precipitate wasseparated by filtration. The precipitate thus obtained was dried, andthereby formazan compound 2 was obtained.

3. Purification of Formazan Compound 2 and Synthesis of TetrazoliumCompound 2

The formazan compound 2 of the above section 2. was dissolved in 10 mLof RO water, and thereby a formazan compound 2 solution was produced. Adisposable column (size: 20 cm×5 cm) was packed with a filler for columnchromatography (manufactured by NACALAI TESQUE, INC., COSMOSIL40C₁₈-PREP), and the disposable column was mounted in a columnpreparative separation system (manufactured by BÜCHI Labortechnik AG,trade name: SEPACORE). The formazan compound 2 solution was purifiedusing this column system. The solvent of a red fraction thus collectedwas removed, and to a solid component thus obtained, 15 mL of methanol,250 μL of 9.6 N HCl, and 5 mL of a 15% ethyl nitrite (CH₃CH₂NO₂)-ethanolsolution were added. The mixture was stirred for 72 hours at roomtemperature (25° C.) in the dark.

4. Collection of Tetrazolium Compound 2

Diethyl ether was added to 5 mL of the reaction solution of the abovesection 3., and thereby tetrazolium compound 2 was precipitated. Theprecipitate was centrifuged, the supernatant was removed, and then theresidue was further washed with diethyl ether. This precipitate wasdried in a draught, and thus tetrazolium compound 2 was obtained.

Example 3: Synthesis of Tetrazolium Compound 3

A compound having the following structure (tetrazolium compound 3) wassynthesized according to the following method.

1. Synthesis of Hydrazone Compound 1

Hydrazone compound 1 was synthesized in the same manner as in 1.Synthesis of hydrazone compound 1 of Example 1.

2. Synthesis of Formazan Compound 3

0.76 g of the hydrazone compound 1 was dissolved in a mixed liquid of 10mL of RO water and 10 mL of DMF, and thereby a hydrazone compound 1solution was produced. 0.351 g of 2-methoxy-4-nitroaniline-5-sulfonicacid sodium salt (manufactured by Goni Chemical Industry Co., Ltd.) wasdissolved in 5.0 mL of RO water. While this solution was maintained at0° C., 280 μL of 9.6 N HCl was added to the solution, a sodium nitritesolution was added dropwise thereto, and diazotization was performed.This diazotized solution was maintained at −20° C., and this solutionwas added dropwise to the hydrazone compound 1 solution. Aftercompletion of the dropwise addition, 300 μL of 10 N NaOH was addeddropwise thereto, the mixture was stirred at room temperature (25° C.)for 2 hours, and thereby a solution including formazan compound 3(formazan compound 3 solution) was produced. The pH of this formazancompound 3 solution was adjusted to neutrality with 9.6 N HCl, and thesolvent was removed. The residue thus obtained was washed withisopropanol, and then a precipitate was separated by filtration. Theprecipitate thus obtained was dried, and thereby formazan compound 3 wasobtained.

3. Purification of Formazan Compound 3 and Synthesis of TetrazoliumCompound 3

The formazan compound 3 of the above section 2. was dissolved in 10 mLof RO water, and thereby a formazan compound 3 solution was produced. Adisposable column (size: 20 cm×5 cm) was packed with a filler for columnchromatography (manufactured by NACALAI TESQUE, INC., COSMOSIL40C₁₈-PREP), and the disposable column was mounted in a columnpreparative separation system (manufactured by BÜCHI Labortechnik AG,trade name: SEPACORE). The formazan compound 3 solution was purifiedusing this column system. The solvent of a red fraction thus collectedwas removed, and to a solid component thus obtained, 15 mL of methanol,250 μL of 9.6 N HCl, and 5 mL of a 15% ethyl nitrite (CH₃CH₂NO₂)-ethanolsolution were added. The mixture was stirred for 72 hours at roomtemperature (25° C.) in the dark.

4. Collection of Tetrazolium Compound 3

Diethyl ether was added to 5 mL of the reaction solution of the abovesection 3., and thereby tetrazolium compound 3 was precipitated. Theprecipitate was centrifuged, the supernatant was removed, and then theresidue was further washed with diethyl ether. This precipitate wasdried, and thus tetrazolium compound 3 was obtained.

Example 4: Synthesis of Tetrazolium Compound 4

A compound having the following structure (tetrazolium compound 4) wassynthesized according to the following method.

1. Synthesis of Hydrazone Compound 1

Hydrazone compound 1 was synthesized in the same manner as in 1.Synthesis of hydrazone compound 1 of Example 1.

2. Synthesis of Formazan Compound 4

0.76 g of the hydrazone compound 1 was dissolved in a mixed liquid of 10mL of RO water and 10 mL of DMF, and thereby a hydrazone compound 1solution was produced. 0.219 g of 2-methoxy-4-nitroaniline (manufacturedby Tokyo Chemical Industry Co., Ltd.) was dissolved in 1.5 mL of ROwater and 5 mL of acetonitrile. While this solution was maintained at 0°C., 280 μL of 9.6 N HCl was added to the solution, a sodium nitritesolution was added dropwise thereto, and diazotization was performed.This diazotized solution was maintained at −20° C., and this solutionwas added dropwise to the hydrazone compound 1 solution. Aftercompletion of the dropwise addition, 300 μL of 10 N NaOH was addeddropwise thereto, the mixture was stirred at room temperature (25° C.)for 2 hours, and thereby a solution including formazan compound 4(formazan compound 4 solution) was produced. The pH of this formazancompound 4 solution was adjusted to neutrality with 9.6 N HCl, and thesolvent was removed. The residue thus obtained was washed with diethylether, and then a precipitate was separated by filtration. Theprecipitate thus obtained was dried, and thereby formazan compound 4 wasobtained.

3. Synthesis of Tetrazolium Compound 4

The formazan compound 4 of the above section 2. was suspended in 15 mLof methanol, and 250 μL of 9.6 N HCl and 5 mL of a 15% ethyl nitrite(CH₃CH₂NO₂)-ethanol solution were added thereto. The mixture was stirredfor 72 hours at room temperature (25° C.) in the dark.

4. Collection of Tetrazolium Compound 4

Diethyl ether was added to 5 mL of the reaction solution of the abovesection 3., and thereby tetrazolium compound 4 was precipitated. Theprecipitate was centrifuged, the supernatant was removed, and then theresidue was further washed with diethyl ether. This precipitate wasdried, and thus tetrazolium compound 4 was obtained.

Example 5: Synthesis of Tetrazolium Compound 5

A compound having the following structure (tetrazolium compound 5) wassynthesized according to the following method.

1. Synthesis of Hydrazone Compound 5

1.213 g of sodium 2-formyl-5-hydroxybenzene sulfonate (manufactured byWako Pure Chemical Industries, Ltd.) and 1.056 g of(6-methoxybenzothiazol-2-yl) hydrazine (also known as2-hydrazino-6-methoxy-1,3-benzothiazole) (manufactured by Santa CruzBiotechnology, Inc.) were dissolved in 43 mL of DMF. This solution washeated and stirred in a water bath at 60° C. for 2 hours under theacidity of acetic acid. After completion of the heating and stirring,the solvent was removed. The residue thus obtained was washed withdiethyl ether, and then a precipitate was separated. This precipitatewas dried, and thereby hydrazone compound 5 was obtained.

2. Synthesis of Formazan Compound 5

1.05 g of the hydrazone compound 5 obtained as described above wasdissolved in a mixed liquid of 20 mL of RO water and 20 mL of DMF, andthereby a hydrazone compound 5 solution was produced. 0.528 g ofo-anisidine-5-sulfonic acid (manufactured by Tokyo Chemical IndustryCo., Ltd.) was suspended in 8.18 mL of RO water, and then 260 μL of 10 NNaOH was added thereto to dissolve the compound. While this solution wasmaintained at 0° C., 560 μL of 9.6 N HCl was added to the solution, asodium nitrite solution was added dropwise thereto, and diazotizationwas performed. This diazotized solution was maintained at −20° C., andthis solution was added dropwise to the hydrazone compound 5 solution.After completion of the dropwise addition, 600 μL of 10 N NaOH was addeddropwise thereto, the mixture was stirred for 2 hours at roomtemperature (25° C.), and thereby a solution including formazan compound5 (formazan compound 5 solution) was produced. The pH of this formazancompound 5 solution was adjusted to neutrality with 9.6 N HCl, and thenthe solvent was removed. The residue thus obtained was washed with ethylacetate, and then a precipitate was separated. This precipitate wasdried, and thus formazan compound 5 was obtained.

3. Synthesis of Tetrazolium Compound 5

The formazan compound 5 obtained in the above section 2. was dissolvedin 10 mL of RO water. A disposable column (size: 20 cm×5 cm) was packedwith a filler for column chromatography (manufactured by NACALAI TESQUE,INC., COSMOSIL 40C₁₈-PREP), and the disposable column was mounted in acolumn preparative separation system (manufactured by BÜCHI LabortechnikAG, trade name: SEPACORE). The formazan compound 5 solution was purifiedusing the above-mentioned column system. The solvent of a red fractionthus collected was removed, and to a solid component thus obtained, 15mL of methanol, 250 μL of 9.6 N HCl, and 5 mL of a 15% ethyl nitrite(CH₃CH₂NO₂)-ethanol solution were added. The mixture was stirred for 72hours at room temperature (25° C.) in the dark.

4. Collection of Tetrazolium Compound 5

Diethyl ether was added to 5 mL of the reaction solution obtained in theabove section 3., and thereby tetrazolium compound 5 was precipitated.The precipitate was centrifuged, the supernatant was removed, and thenthe residue was further washed with diethyl ether. A precipitate thusobtained was dried in a draught, and thus tetrazolium compound 5 wasobtained (120 mg, yield: 7.6% by mass).

Example 6: Synthesis of Tetrazolium Compound 6

A compound having the following structure (tetrazolium compound 6) wassynthesized according to the following method.

1. Synthesis of Hydrazone Compound 6

1.213 g of sodium 5-formyl-2-hydroxybenzene sulfonate (also known as4-formyl-1-phenol-2-sulfonic acid sodium salt) (manufactured by T&WGROUP) and 1.056 g of (6-methoxybenzothiazol-2-yl)hydrazine (also knownas 2-hydrazino-6-methoxy-1,3-benzothiazole) (manufactured by Santa CruzBiotechnology, Inc.) were dissolved in 43 mL of DMF. This solution washeated and stirred in a water bath at 60° C. for 2 hours under theacidity of acetic acid. After completion of the heating and stirring,the solvent was removed. The residue thus obtained was washed withdiethyl ether, and then a precipitate was separated by centrifuge. Theprecipitate thus obtained was dried, and thus hydrazone compound 6 wasobtained.

2. Synthesis of Formazan Compound 6

1.05 g of the hydrazone compound 6 obtained as described above wasdissolved in 20 mL of RO water and 20 mL of DMF, and thereby a hydrazonecompound 6 solution was produced. 0.528 g of o-anisidine-5-sulfonic acid(manufactured by Tokyo Chemical Industry Co., Ltd.) was suspended in8.18 mL of RO water, and 260 μL of 10 N NaOH was added thereto todissolve the compound. While this solution was maintained at 0° C., 560μL of 9.6 N HCl was added to the solution, a sodium nitrite solution wasadded dropwise thereto, and diazotization was performed. While thisdiazotized solution was maintained at −20° C., this solution was addeddropwise to the hydrazone compound 6 solution. After completion of thedropwise addition, 600 μL of 10 N NaOH was added dropwise thereto, themixture was stirred at room temperature (25° C.) for 2 hours, andthereby a solution including formazan compound 6 (formazan compound 6solution) was produced. The pH of this formazan compound 6 solution wasadjusted to 6.8 with 9.6 N HCl, and the solvent was removed. The residuethus obtained was washed with ethyl acetate, and then a precipitate wasseparated by centrifugation. The precipitate thus obtained was dried,and thus formazan compound 6 was obtained.

3. Synthesis of Tetrazolium Compound 6

The formazan compound 6 obtained in the above section 2. was added to 15mL of methanol, 250 μL of 9.6 N HCl, and 5 mL of a 15% ethyl nitrite(CH₃CH₂NO₂)-ethanol solution, and the mixture was stirred for 72 hoursat room temperature (25° C.) in the dark.

4. Collection of Tetrazolium Compound 6

Diethyl ether was added to the reaction solution obtained in the abovesection 3., and thereby tetrazolium compound 6 was precipitated. Theprecipitate was centrifuged, the supernatant was removed, and then theresidue was further washed with diethyl ether. A precipitate thusobtained was dried, and thus tetrazolium compound 6 was obtained.

Example 7: Synthesis of Tetrazolium Compound 7

A compound having the following structure (tetrazolium compound 7) wassynthesized according to the following method.

1. Synthesis of Hydrazone Compound 7

25.0 g of 2-sulfobenzaldehyde sodium salt (manufactured by TokyoChemical Industry Co., Ltd.) and 22.6 g of p-hydrazinobenzene sulfonicacid 0.5 hydrate (manufactured by Tokyo Chemical Industry Co., Ltd.)were dissolved in 250 mL of RO water, and 11.8 g of sodium acetate wasadded thereto. The mixture was heated and stirred in a water bath at 60°C. for 2 hours. After completion of the heating and stirring, thesolvent was removed. The residue thus obtained was washed with methanol,and then a precipitate was separated by filtration. The precipitate thusobtained was dried, and thus hydrazone compound 7 was obtained.

2. Synthesis of Formazan Compound 7

0.6 g of the hydrazone compound 7 obtained as described above wasdissolved in 20 mL of RO water, and thereby a hydrazone compound 7solution was produced. 0.234 g of 2-amino-6-methoxybenzothiazole(manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in 1.5mL of RO water and 5 mL of acetonitrile. While this solution wasmaintained at 0° C., 280μ of 9.6 N HCl was added to the solution, asodium nitrite solution was added dropwise thereto, and diazotizationwas performed. This diazotized solution was maintained at −20° C., andthis solution was added dropwise to the hydrazone compound 7 solution.After completion of the dropwise addition, 300 μL of 10 N NaOH was addeddropwise thereto, the mixture was stirred for 2 hours at roomtemperature (25° C.), and thereby a solution including formazan compound7 (formazan compound 7 solution) was produced. The pH of this formazancompound 7 solution was adjusted to neutrality with 9.6 N HCl, and thesolvent was removed. The residue thus obtained was washed withisopropanol, and then a precipitate was separated by filtration. Thisprecipitate was dried, and thus formazan compound 7 was obtained.

3. Synthesis of Tetrazolium Compound 7

The formazan compound 7 of the above section 2. was added to 15 mL ofmethanol, 250 μL of 9.6 N HCl, and 5 mL of a 15% ethyl nitrite(CH₃CH₂NO₂)-ethanol solution, and the mixture was stirred for 72 hoursat room temperature (25° C.) in the dark.

4. Collection of Tetrazolium Compound 7

Diethyl ether was added to 5 mL of the reaction solution of the abovesection 3., and thereby tetrazolium compound 7 was precipitated. Theprecipitate was centrifuged, the supernatant was removed, and then theresidue was further washed with diethyl ether. A precipitate thusobtained was dried, and thus tetrazolium compound 7 was obtained.

Example 8: Synthesis of Tetrazolium Compound 8

A compound having the following structure (tetrazolium compound 8) wassynthesized according to the following method.

Tetrazolium compound 8 was obtained in the same manner as in Example 1,except that in regard to 1. Synthesis of hydrazone compound 1 of Example1, disodium 4-formylbenzene-1,3-disulfonate (manufactured by TokyoChemical Industry Co., Ltd.) was changed to5-formylbenzene-1,3-disulfonic acid disodium salt) (manufactured byTokyo Chemical Industry Co., Ltd.); and in regard to the synthesis offormazan compound 1, the amine was changed from p-anisidine-3-sulfonicacid (manufactured by Tokyo Chemical Industry Co., Ltd.) too-anisidine-3-sulfonic acid.

Example 9: Synthesis of Tetrazolium Compound 9

A compound having the following structure (tetrazolium compound 9) wassynthesized according to the following method.

Tetrazolium compound 9 was obtained in the same manner as in Example 1,except that in regard to 1. Synthesis of hydrazone compound 1 of Example1, disodium 4-formylbenzene-1,3-disulfonate (manufactured by TokyoChemical Industry Co., Ltd.) was changed to disodium5-formylbenzene-1,3-disulfonate.

Example 10: Synthesis of Tetrazolium Compound 10

A compound having the following structure (tetrazolium compound 10) wassynthesized according to the following method.

Tetrazolium compound 10 was obtained in the same manner as in Example 1,except that in regard to the synthesis of formazan compound 1 of Example1, the amine was changed from p-anisidine-3-sulfonic acid (manufacturedby Tokyo Chemical Industry Co., Ltd.) to 4-amino-3-methoxybenzoic acid(manufactured by Tokyo Chemical Industry Co., Ltd.).

Example 11: Synthesis of Tetrazolium Compound 11

A compound having the following structure (tetrazolium compound 11) wassynthesized according to the following method.

1. Synthesis of Hydrazone Compound 11

4.65 g (0.015 mol) of 5-formylbenzene-1,3-disulfonic acid disodium salt(manufactured by Tokyo Chemical Industry Co., Ltd.) and 2.93 g (0.015mol) of (6-methoxybenzothiazol-2-yl)hydrazine (also known as2-hydrazino-6-methoxy-1,3-benzothiazole) (manufactured by Santa CruzBiotechnology, Inc.) were dissolved in a mixed liquid of 50 mL of DMFand 50 mL of RO water. 860 μL of acetic acid was added to this solution,and the mixture was heated with stirring in a water bath (60° C.) for 2hours. After completion of the heating and stirring, the solvent wasremoved, and a residue was obtained. This residue was washed withdiethyl ether, and then a precipitate was separated by filtration. Thisprecipitate was dried, and thereby hydrazone compound 11 was obtained.

2. Synthesis of Formazan Compound 11

1.4 g of the hydrazone compound 11 of the above section 1. was dissolvedin a mixed liquid of 10 mL of RO water and 5 mL of DMF, and thus ahydrazone compound 11 solution was produced. 0.334 g of3-amino-4-methoxybenzoic acid (manufactured by Tokyo Chemical IndustryCo., Ltd.) was dissolved in a mixed liquid of 1 mL of RO water and 5 mLof DMF. This solution was maintained at 0° C., and 400 μL of 9.6 N HClwas added thereto. Subsequently, a sodium nitrite solution (0.152 g wasdissolved in 1 mL) was further added to the mixture, and diazotizationwas performed. This diazotized solution was maintained at −20° C., andthis solution was added to the hydrazone compound 11 solution. Next, 600μL of 10 N NaOH was added thereto, the mixture was stirred for 2 hoursat room temperature, and thereby a solution including the formazancompound 11 (formazan compound 11 solution) was produced. The pH of thisformazan compound 11 solution was adjusted to neutrality with 9.6 N HCl,and the solution was concentrated. 10 mL of RO water was added to theconcentrated formazan compound 11 to dissolve the compound, and thus aformazan compound 11 solution was produced.

3. Purification of Formazan Compound 11 and Synthesis of TetrazoliumCompound 11

A disposable column (size: 20 cm×5 cm) was packed with a filler forcolumn chromatography (manufactured by NACALAI TESQUE, INC., COSMOSIL40C₁₈-PREP), and the disposable column was mounted in a columnpreparative separation system (manufactured by BÜCHI Labortechnik AG,trade name: SEPACORE). The formazan compound 11 solution was purifiedusing this column system. The solvent of a red fraction thus collectedwas removed, and to a solid component thus obtained, 100 mL of methanol,400 μL of 9.6 N HCl, and 5 mL of a 15% ethyl nitrite (CH₃CH₂NO₂)-ethanolsolution were added. The mixture was stirred for 48 hours at roomtemperature in the dark.

4. Collection of Tetrazolium Compound 11

The above-described solution was dried with an evaporator. Next, 10 mLof RO water was added to the residue, the mixture was neutralized with 1M Na₂CO₃, and the mixture was dissolved. This was purified by a SEPACOREpreparative separation system, and an orange-colored fraction wascollected. The solvent was removed from this fraction with anevaporator, and a solid content was obtained. 5 mL of methanol was addedto this solid content to dissolve the solid, and then 50 mL of diethylether was added thereto, and there by a precipitate was obtained. Thisprecipitate was dried, and thus tetrazolium compound 11 was obtained.

Example 12: Synthesis of Tetrazolium Compound 12

A compound having the following structure (tetrazolium compound 12) wassynthesized according to the following method.

1. Synthesis of Hydrazone Compound 1

Hydrazone compound 1 was synthesized in the same manner as in 1.Synthesis of hydrazone compound 1 of Example 1.

2. Synthesis of Formazan Compound 12

1.4 g of hydrazone compound 1 was added to a mixed solution of 10 mL ofRO water and 5 mL of DMF, and thereby a hydrazone compound 1 solutionwas produced. 0.334 g of 5-amino-2-methoxybenzoic acid (manufactured byTokyo Chemical Industry Co., Ltd.) was added to 1 mL of RO water and 5mL of DMF to dissolve the compound. This solution was maintained at 0°C., and 400 μL of 9.6 N HCl was added thereto. Subsequently, a sodiumnitrite solution (0.152 g dissolved in 1 mL; Wako Pure ChemicalIndustries, Ltd.) was further added thereto, and diazotization wasperformed. This diazotized solution was maintained at −20° C., and thissolution was added to a hydrazone compound 1 solution. Subsequently, 600μL of 10 N NaOH was added thereto, subsequently the mixture was stirredat room temperature for 2 hours, and thereby, a solution includingformazan compound 12 (formazan compound 12 solution) was produced. ThepH of the formazan compound 12 solution was adjusted to neutrality with9.6 N HCl, the solvent was removed, and thereby formazan compound 12 wasobtained.

3. Purification of Formazan Compound 12 and Synthesis of TetrazoliumCompound 12

The formazan compound 12 of the above section 2. was dissolved in 10 mLof RO water, and thereby a formazan compound 12 solution was produced. Adisposable column (size: 20 cm×5 cm) was packed with a filler for columnchromatography (manufactured by NACALAI TESQUE, INC., COSMOSIL40C₁₈-PREP), and the disposable column was mounted in a columnpreparative separation system (manufactured by BÜCHI Labortechnik AG,trade name: SEPACORE). The formazan compound 12 solution was purifiedusing this column system. The solvent of a red fraction thus collectedwas removed, and to a solid component thus obtained, 100 mL of methanol,400 μL of 9.6 N HCl, and 15 mL of a 15% ethyl nitrite(CH₃CH₂NO₂)-ethanol solution were added. The mixture was stirred for 48hours at room temperature in the dark.

4. Collection of Tetrazolium Compound 12 and Repurification with Column

The solution of the above section 3. was dried, and then 10 mL of ROwater was added thereto. The solution was neutralized with 1 M Na₂CO₃ todissolve the compound. This was purified with a column preparativeseparation system (manufactured by BÜCHI Labortechnik AG, trade name:SEPACORE), and an orange-colored fraction was collected. The solvent ofthis fraction was removed with an evaporator, and a solid content wasobtained. 5 mL of methanol was added to this solid content to dissolvethe solid, and then 50 mL of diethyl ether was added thereto. Thereby, aprecipitate was obtained. This precipitate was dried, and thustetrazolium compound 12 was obtained (300 mg, yield: 17%).

Example 13: Synthesis of Tetrazolium Compound 13

A compound having the following structure (tetrazolium compound 13) wassynthesized according to the following method.

1. Synthesis of Hydrazone Compound 13

1.38 g of disodium 4-formylbenzene-1,3-disulfonate (manufactured byTokyo Chemical Industry Co., Ltd.) and 0.9831 g of6-hydrazino[1,3]dioxolo[4,5-f][1,3]benzothiazole (manufactured by MatrixScientific) were suspended in 10 mL of DMF and 10 mL of RO water. Thissuspension was stirred in a water bath (60° C.) for 90 minutes under theacidity of acetic acid. After completion of the heating and stirring,the suspension was concentrated (0 Torr, 60° C.). This residue waswashed two times with diethyl ether, and then a precipitate waspreoperatively separated. This precipitate was dried, and therebyhydrazone compound 13 was obtained.

2. Synthesis of Formazan Compound 13

0.1 g of the hydrazone compound 13 was dissolved in a mixed liquid of 1mL of RO water and 1 mL of DMF, and thereby a hydrazone compound 13solution was produced. This hydrazone compound 13 solution wasmaintained at 0° C., and 0.04 g of o-anisidine-5-sulfonic acid(manufactured by Tokyo Chemical Industry Co., Ltd.) was suspended in 1mL of RO water. 20 μL of 10 N NaOH was added thereto, and the compoundwas dissolved. This solution was maintained at 0° C., 40 μL of 10 N HClwas added to the solution, and then a sodium nitrite solution wasfurther added thereto. Thereby, diazotization was performed. Thisdiazotized solution was maintained at −20° C., and this solution wasadded dropwise to the hydrazone compound 13 solution. Subsequently, 40μL of 10 N NaOH was added thereto, the mixture was stirred at 4° C., andthereby, a solution including formazan compound 13 (formazan compound 13solution) was produced. The pH of this formazan compound 13 solution wasadjusted to neutrality with 9.6 N HCl, and the solvent was removed. Theresidue thus obtained was washed with isopropanol, and then aprecipitate was separated by filtration. The precipitate thus obtainedwas dried, and thereby formazan compound 13 was obtained.

The formazan compound thus obtained was purified in the same manner asin Example 1, and a tetrazolium compound was synthesized and collectedin the same manner. Thus, tetrazolium compound 13 was synthesized.

Examples 14 to 16: Synthesis of Tetrazolium Compounds 14 to 16

Compounds having the following structures (tetrazolium compounds 14 to16) were synthesized according to the following method.

1. Synthesis of Hydrazone Compounds 14 to 16

Hydrazone compounds 14 to 16 were synthesized in the same manner as inExample 13, except that various reagents and various solvents were addedas described below.

TABLE 1 Amount Amount of of Acetic RO addition addition acid waterHydrazine※1 (g) Aldehyde※2 (g) (μL) DMF (mL) (mL) Example 6-Hydrazino[1,0.9831 Disodium 1.38 256 10 mL 10 mL 13 3]dioxolo[4,5-f][1,4-Formylbenzene-1, 3]benzothiazole 3-disulfonate Example 2-Hydrazino-6,0.9666 1.27 236 14 7-dihydro[1, 4]dioxino[2,3-f][1, 3]benzothiazoleExample 2-Hydrazino-6-methoxy-1, 0.984 1.38 256 15 3-benzothiazoleExample 2-Hydrazino-5, 0.9767 1.27 236 16 6-dimethoxy-1, 3-benzothiazole※1All manufactured by Matrix Scientific ※2All manufactured by TokyoChemical Industry Co., Ltd.

2. Synthesis of Formazan Compounds 14 to 16

Synthesis of formazan compounds 14 to 16 was performed in the samemanner as in Example 13, except that various reagents and varioussolvents were added as described below.

TABLE 2 Amount of Amount of addition addition (mg) Amine※3 (mg) Example13 Hydrazone 100 o-anisidin- 40 compound 13 5-sulfonic Example 14Hydrazone 103 acid compound 14 Example 15 Hydrazone 100 compound 15Example 16 Hydrazone 104 compound 16

The formazan compounds thus obtained were purified in the same manner asin Example 1, and tetrazolium compounds synthesized and collected in thesame manner. Thus, tetrazolium compounds 14 to 16 were synthesized.

Example 17: Synthesis of Tetrazolium Compound 17

A compound having the following structure (tetrazolium compound 17) wassynthesized according to the following method.

1. Synthesis of Hydrazone Compound 17

1.38 g (4.46 mmol) of disodium 4-formylbenzene-1,3-disulfonate(manufactured by Tokyo Chemical Industry Co., Ltd.) and 0.98 g (4.46mmol) of 6-hydrazino[1,3]dioxolo[4,5-f][1,3]benzothiazole (manufacturedby Matrix Scientific) were dissolved in a mixed liquid of 10 mL of DMFand 10 mL of RO water. 256 μL of acetic acid was added to this solution,and the mixture was stirred in a water bath (60° C.) for 2 hours. Aftercompletion of the heating and stirring, the solvent was removed, and aresidue was obtained. This residue was stirred for one hour in diethylether, the residue was washed, and then a precipitate was separated byfiltration. This precipitate was dried, and thus hydrazone compound 17was obtained.

2. Synthesis of Formazan Compound 17

1.81 g of hydrazone compound 17 of the above section 1. was dissolved ina mixed liquid of 15 mL of RO water and 15 mL of DMF, and thereby, ahydrazone compound 17 solution was produced. 0.528 g ofp-anisidine-3-sulfonic acid (manufactured by Tokyo Chemical IndustryCo., Ltd.) was suspended in 8.18 mL of RO water, 260 μL of 10 N NaOH wasfurther added thereto to dissolve the compound. This solution wasmaintained at 0° C., and 560 μL of 9.6 N HCl was added to the solution.A sodium nitrite solution (0.194 g was dissolved in 1 mL; Wako PureChemical Industries, Ltd.) was added thereto, and diazotization wasperformed. This diazotized solution as maintained at −20° C., and thissolution was added dropwise to the hydrazone compound 17 solution. Aftercompletion of the dropwise addition, 600 μL of 10 N NaOH was addedthereto, and the mixture was stirred at −20° C. Subsequently, thesolvent of this solution was removed, the residue was dried, andthereby, formazan compound 17 was obtained.

3. Purification of Formazan Compound 17 and Synthesis of TetrazoliumCompound 17

15 mL of RO water was added to the formazan compound 17 of the abovesection 2., and thereby, a formazan compound 17 solution was produced. Adisposable column (size: 20 cm×5 cm) was packed with a filler for columnchromatography (manufactured by NACALAI TESQUE, INC., COSMOSIL40C₁₈-PREP), and the disposable column was mounted in a columnpreparative separation system (manufactured by BÜCHI Labortechnik AG,trade name: SEPACORE). The formazan compound 17 solution was purifiedusing this column system, and a red fraction was collected. The solventof this fraction was removed, and to the solid content thus obtained, 15mL of methanol, 250 μL of 9.6 N HCl, and 5 mL of a 15% ethyl nitrite(CH₃CH₂NO₂)-ethanol solution were added. The mixture was stirred for 72hours at room temperature in the dark.

4. Purification and Collection of Tetrazolium Compound 17

The solution obtained in the above section 3. was dried to solid underreduced pressure, and a residue was obtained. 15 mL of RO water wasadded to this residue, and the residue was dissolved. This solution wasmounted in a column preparative separation system (manufactured by BÜCHILabortechnik AG, trade name: SEPACORE), and an orange-colored fractionincluding tetrazolium compound 17 was collected. This fraction was driedto solid under reduced pressure, and a purification product includingthe tetrazolium compound 17 was obtained. 5 mL of methanol was added tothis purification product, and the purification product was dissolvedtherein. Subsequently, diethyl ether was added thereto under stirring,and thereby tetrazolium compound 17 was precipitated. This liquid wascentrifuged, and the supernatant was removed. The remaining precipitate(tetrazolium compound 17) was dried, and thus tetrazolium compound 17was obtained (620 mg, yield: 26% by mass, results of purity analysis byUPLC: 96.1%).

Example 18: Synthesis of Tetrazolium Compound 18

A compound having the following structure (tetrazolium compound 18) wassynthesized according to the following method.

1. Synthesis of Hydrazone Compound 18

1.27 g (4.11 mmol) of disodium 4-formylbenzene-1,3-disulfonate(manufactured by Tokyo Chemical Industry Co., Ltd.) and 0.966 g (4.11mmol) of 2-hydrazino-5,6-dimethoxy-1,3-benzothiazole (manufactured byMatrix Scientific) were added to a mixed liquid of 10 mL of DMF and 10mL of RO water to dissolve the compounds. 236 μL of acetic acid wasadded to this solution, and the mixture was stirred in a water bath (60°C.) for 2 hours. After completion of the heating and stirring, themixture was dried to solid under reduced pressure, and a residue wasobtained. This residue was washed with diethyl ether under stirring, andthen a precipitate was preoperatively separated. This precipitate wasdried, and thus hydrazone compound 18 was obtained.

2. Synthesis of Formazan Compound 18

1.81 g of formazan compound 18 was added to a mixed liquid of 15 mL ofRO water and 15 mL of DMF, and the compound was dissolved therein. 0.528g of p-anisidine-3-sulfonic acid (manufactured by Tokyo ChemicalIndustry Co., Ltd.) was suspended in 8.18 mL of RO water, and then 260μL of 10 N NaOH was added to this suspension to dissolve the compound.This solution was maintained at 0° C., and 560 μL of 9.6 N HCl was addedthereto. A sodium nitrite solution (0.194 g was dissolved in 1 mL; WakoPure Chemical Industries, Ltd.) was added dropwise to the mixture, anddiazotization was performed. This diazotized solution was maintained at−20° C., and this solution was added dropwise to the hydrazone solution.After completion of the dropwise addition, 600 μL of 10 N NaOH was addeddropwise thereto, and the mixture was stirred for one hour at −20° C.Thus, a formazan compound 18 solution was obtained. This formazancompound 18 solution was dried to solid under reduced pressure, andthereby a residue was obtained. This residue was washed withisopropanol, and then a precipitate was separated by filtration. Thisprecipitate was dried, and thus formazan compound 18 was obtained.

3. Purification of Formazan Compound 18 and Synthesis of TetrazoliumCompound 18

Formazan compound 18 of the above section 2. was added to 15 mL of ROwater to dissolve the compound, and a formazan compound 18 solution wasproduced. A disposable column (size: 20 cm×5 cm) was packed withCOSMOSIL 40C₁₈-PREP (NACALAI TESQUE, INC.), and the disposable columnwas mounted in a column preparative separation system (manufactured byBÜCHI Labortechnik AG, trade name: SEPACORE). A fraction including theformazan compound 18 solution was collected using this column system.The solvent of this fraction was removed, and to the solid content thusobtained, 100 mL of methanol, 20 mL of a 15% ethyl nitrite-ethanolsolution (manufactured by Tokyo Chemical Industry Co., Ltd.), and 0.5 mLof 9.6 N HCl were added. The mixture was stirred for 48 hours at roomtemperature in the dark, and thus a solution including the tetrazoliumcompound 18 was obtained.

4. Collection of Tetrazolium Compound 18

The solution obtained in the above section 3. was dried to solid underreduced pressure, and a crude purification product of the tetrazoliumcompound 18 was obtained. 15 mL of RO water was added to this crudepurification product, and the purification product was dissolvedtherein. This solution was mounted in a column preparative separationsystem (manufactured by BÜCHI Labortechnik AG, trade name: SEPACORE).The tetrazolium compound 18 was purified using this column system. Afraction solution thus collected was dried to solid under reducedpressure, and thus a residue was obtained. This residue was suspended in10 mL of methanol. Furthermore, 100 mL of diethyl ether was added to thesuspension under stirring, and thereby the tetrazolium compound 18 wasprecipitated. This liquid was centrifuged, the supernatant was removed,and thereby a precipitate was washed. The remaining precipitate(tetrazolium compound 18) was dried (yield amount: 1.2 g, yield: 51%,purity analysis by UPLC: 99.2%).

Example 19: Synthesis of Tetrazolium Compound 19

A compound having the following structure (tetrazolium compound 19) wassynthesized according to the following method.

1. Synthesis of Hydrazone Compound 19

1.27 (4.11 mmol) of disodium 4-formylbenzene-1,3-disulfonate(manufactured by Tokyo Chemical Industry Co., Ltd.) and 0.976 g (4.11mmol) of 2-hydrazino-6,7-dihydro[1,4]dioxino[2,3-f][1,3]benzothiazole(manufactured by Matrix Scientific) were dissolved in a mixed liquid of10 mL of DMF and 10 mL of RO water. 236 μL of acetic acid was added tothis solution, and the mixture was heated and stirred in a water bath(60° C.) for 2 hours. After completion of the heating and stirring, themixture was dried to solid under reduced pressure, and a residue wasobtained. Diethyl ether was added to this residue, and the mixture wasstirred and washed for one hour. Subsequently, a precipitate waspreparatively separated by centrifugation. Subsequently, the precipitatewas dried overnight under reduced pressure, and thus hydrazone compound19 was obtained.

2. Synthesis of Formazan Compound 19

1.81 g of hydrazone compound 19 was dissolved in a mixed liquid of 10 mLof RO water and 10 mL of DMF, and thereby a hydrazone compound 19solution was produced. 0.528 g of p-anisidine-3-sulfonic acid(manufactured by Tokyo Chemical Industry Co., Ltd.) was suspended in8.18 mL of RO water. 260 μL of 10 N NaOH was added to this suspension,and the compound was dissolved therein. This solution was maintained at0° C., and 560 μL of 9.6 N HCl was added thereto. A sodium nitritesolution (0.194 g was dissolved in 1 mL) was added dropwise to themixture, and diazotization was performed. This diazotized solution wasmaintained at −20° C., and this solution was added dropwise to thehydrazone compound 19 solution. After completion of the dropwiseaddition, 600 μL of 10 N NaOH was added thereto, and the mixture wasstirred for one hour at −20° C. Thus, a solution including formazancompound 19 was obtained. This solution was dried to solid under reducedpressure, and a residue was obtained. This residue was washed withisopropanol, and then a precipitate was separated by filtration. Thisprecipitate was dried, and thus formazan compound 19 was obtained.

3. Purification of Formazan Compound 19 and Synthesis of TetrazoliumCompound 19

15 mL of RO water was added to the formazan compound 19 of the abovesection 2., and the compound was dissolved therein. Thus, a formazancompound 19 solution was produced. A disposable column (size: 20 cm×5cm) was packed with COSMOSIL 40C₁₈-PREP (NACALAI TESQUE, INC.), and thedisposable column was mounted in a column preparative separation system(manufactured by BÜCHI Labortechnik AG, trade name: SEPACORE). Theformazan compound 19 solution was purified using this column system. Thesolvent of a red fraction thus collected was dried to solid underreduced pressure, and thereby, a residue was obtained. To this residue,100 mL of methanol, 20 mL of a 15% ethyl nitrite-ethanol solution(manufactured by Tokyo Chemical Industry Co., Ltd.), and 0.5 mL of 9.6 NHCl were added, and the mixture was stirred for 48 hours at roomtemperature in the dark.

4, Collection of Tetrazolium Compound 19

The solution obtained in the above section 3. was dried to solid underreduced pressure, and a residue was obtained. 15 mL of RO water wasadded to a residue including this tetrazolium compound 19, and thecompound was dissolved therein. This solution was purified with aSEPACORE preparative separation system. A fraction thus collected wasdried to solid under reduced pressure, and a residue including thetetrazolium compound 19 was obtained. 5 mL of methanol was added to thisresidue to dissolve the residue. While this solution was stirred, 100 mLof diethyl ether was added thereto, and the tetrazolium compound 19 wasprecipitated. This was centrifuged, the supernatant was removed, andthereby the precipitate was washed. The remaining precipitate(tetrazolium compound 19) was dried, and thus, tetrazolium compound 19was obtained (yield amount: 0.93 g, yield: 40% by mass, purity analysisby UPLC: 96.4%).

Example 20: Synthesis of Tetrazolium Compound 20

A compound having the following structure (tetrazolium compound 20) wassynthesized according to the following method.

1. Synthesis of Hydrazone Compound 20

1.38 g (4.46 mmol) of disodium 4-formylbenzene-1,3-disulfonate(manufactured by Tokyo Chemical Industry Co., Ltd.) and 0.98 g (4.46mmol) of (6-ethoxy-benzothiazol-2-yl)hydrazine (manufactured by MatrixScientific) were dissolved in a mixed liquid of 10 mL of DMF and 10 mLof RO water, and thereby a hydrazone compound 20 was produced. 256 μL ofacetic acid was added to this solution, and the mixture was heated andstirred in a water bath (60° C.) for 2 hours. After completion of theheating and stirring, the solvent was removed, and a residue wasobtained. 100 mL of diethyl ether was added to this residue, the mixturewas stirred for one hour, and then a precipitate was preoperativelyseparated by centrifugation. The precipitate was left to stand for 2hours in a draught, and subsequently the precipitate was dried overnightunder reduced pressure. Thus, hydrazone compound 20 was obtained.

2. Synthesis of Formazan Compound 20

1.81 g of hydrazone compound 20 was dissolved in a mixed liquid of 15 mLof RO water and 15 mL of DMF, and thereby a hydrazone compound 20solution was produced. Apart from this, 0.528 g ofp-anisidine-3-sulfonic acid (manufactured by Tokyo Chemical IndustryCo., Ltd.) was suspended in 8.18 mL of RO water, 260 μL of 10 N NaOH wasadded to the suspension, and the compound was dissolved therein. Thissolution was maintained at 0° C., and 560 μL of 9.6 N HCl was addedthereto (turbid, dissolved when nitrous acid was added). A sodiumnitrite solution (0.194 g was dissolved in 1 mL) was added dropwisethereto, and diazotization was performed. This diazotization solutionwas maintained at −20° C., and this solution was added dropwise to thehydrazone compound 20 solution. After completion of the dropwiseaddition, 600 μL of 10 N NaOH was added dropwise thereto, the mixturewas stirred for one hour at −20° C., and thereby a formazan compound 20solution was obtained. The solvent of this formazan compound 20 solutionwas removed with an evaporator. The residue thus obtained was washedwith isopropanol, and then a precipitate was separated by filtration.This precipitate was dried, and thus formazan compound 20 was obtained.

3. Purification of Formazan Compound 20 and Synthesis of TetrazoliumCompound 20

The formazan compound 20 of the above section 2. was dissolved in 15 mLof RO water, and thereby a formazan compound 20 solution was produced. Adisposable column (size: 20 cm×5 cm) was packed with a filler for columnchromatography (manufactured by NACALAI TESQUE, INC., COSMOSIL40C₁₈-PREP), and the disposable column was mounted in a columnpreparative separation system (manufactured by BÜCHI Labortechnik AG,trade name: SEPACORE). A fraction including the formazan compound 20solution was collected using this column system. The solvent of thisfraction was removed, and to a solid content thus obtained, 100 mL ofmethanol, 20 mL of 15% ethyl nitrite-ethanol solution (manufactured byTokyo Chemical Industry Co., Ltd.), and 0.5 mL of 9.6 N HCl were added.The mixture was stirred for 48 hours at room temperature in the dark.

4. Collection of Tetrazolium Compound 20

The solution of the above section 3. was dried to solid under reducedpressure, and a crude purification product of tetrazolium compound 20was obtained. 15 mL of RO water was added to this crude purificationproduct, and the purification product was dissolved therein. Thissolution was purified with a SEPACORE preparative separation system. Thesolvent of a fraction solution thus collected was removed, and a residueincluding tetrazolium compound 20 was obtained. This residue wasdissolved in 5 mL of methanol. Subsequently, diethyl ether was addedthereto with stirring, and thereby tetrazolium compound 20 wasprecipitated. This was centrifuged, a precipitate was washed, and thenthe precipitate was preoperatively separated. This precipitate wasdried, and thus tetrazolium compound 20 was obtained (amount of yield:1.4 g, yield: 60%, result of purity analysis by UPLC: 100.0%).

For the tetrazolium compounds 1 to 20 obtained in Examples 1 to 20described above, and comparative compounds 1 to 5 (Comparative Examples1 to 5) having the following structures, the maximum absorptionwavelength (λmax), the chelation rate, sensitivity, and water-solubilitywere evaluated according to the methods described below, and the resultsare presented in the following Table 3.

(Evaluation of Chelation Rate and Maximum Absorption Wavelength (λMax))

10 mM aqueous solution of MOPS was added to each of the formazancompounds so as to obtain a final concentration of 50 to 200 mM, andthereby 100 μL of a sample was produced. At this time, all of thesamples were red-brown in color. Apart from this, a 1 M aqueous solutionof nickel ion was produced.

10 μL of the aqueous solution of nickel ion thus produced was added tothe sample described above, and while the mixture was rapidly stirred, achange in the color tone was observed. The time taken to visuallyconfirm color development after addition of the aqueous nickel solutionwas measured. In a case in which the time was one minute or less, thechelation rate was rated as “◯”; and in a case in which the time waslonger than one minute, the chelation rate was rated as “×”. InComparative Examples 2 and 5, since no change in the color tone wasobserved, it was considered that no chelate was formed. Thus, in Table3, it was indicated that “No chelation”.

For mixed solutions of various compounds in an aqueous solution offormazan and an aqueous solution of nickel, the spectra were measuredwith a spectrophotometer (measurement cell length: 10 mm) (n=1). Basedon each of the spectra, the maximum absorption wavelength (λmax) (nm) ofeach of the compounds in formazan was determined. The respective resultsare presented in Table 3. As an example, the spectrum of a chelatecompound of formazan compound 1 and nickel ion is shown in FIG. 1. FromFIG. 1, it is understood that the maximum absorption wavelength (λmax)of the chelate compound of formazan compound 1 and nickel ion is 630 nm.

FIG. 19 shows the spectra of Ni²⁺ chelate compounds of formazan producedfrom tetrazolium compounds 1 and 13 to 18. The absorbance at the maximumabsorption wavelength is denoted as 100%. The maximum absorptionwavelength of formazan compound 17 or 18 having a4-methoxy-5-sulfophenyl group and having a q value of 2 was shiftedtoward the higher wavelength side.

(Evaluation of Sensitivity)

To 0.02 mmol of each compound, 75 μL of RO water and 100 μL of a 0.5 MMOPS solution (pH 7.2) were added to dissolve the compound, and therebya sample was produced. As a control, 85 μL of RO water and 100 μL of a0.5 mMOPS solution (pH 7.2) were added to 11.6 mg of2-benzothiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylcarbamoyl)phenyl]-2H-tetrazolium(WST-4) to dissolve the compound, and thereby a control sample wasproduced.

Separately, 100 μL of RO water was added to 7 mg of a glucosedehydrogenase that uses flavin adenine dinucleotide (FAD) as a coenzyme(GDH-FAD) (manufactured by TOYOBO CO., LTD., product No.: GLD-351), theenzyme was dissolved, and thus a GDH solution was produced. Furthermore,200 μL of RO water was added to 3.5 mg of 1-methoxy-5-methylphenaziummethyl sulfate (m-PMS) (manufactured by DOJINDO LABORATORIES), and thecompound was dissolved therein. Thus, an s-PMS solution was produced. 1mL of RO water was added to 129 mg of nickel chloride to dissolve thesalt, and thus a nickel solution was produced.

To 175 μL of each sample, 10 μL of the GDH solution, 5 μL of the m-PMSsolution, and 10 μL of the nickel solution, all of which had beenproduced as described above, were added, and the mixture was called areaction solution. Furthermore, to 175 μL of a control sample, 10 μL ofthe GDH solution and 5 μL of the m-PMS solution, both of which had beenproduced as described above, and 10 μL of RO water were added, and themixture was called a control reaction solution.

40 μL of the reaction solution was added to 10 μL each of an aqueousglucose solution at a concentration of 125 mg/dL, an aqueous glucosesolution at a concentration of 250 mg/dL, and an aqueous glucosesolution at a concentration of 1,000 mg/dL, and the mixtures wereallowed to develop color. The spectra were measured with aspectrophotometer (measurement cell length: 50 μm) (n=1). For each ofthe spectra, the absorbance at the maximum absorption wavelength (λmax)of each of the compounds determined as described above was measured, andthe glucose concentration in the colored solution and the absorbancewere plotted on the horizontal axis and the vertical axis, respectively.Thus, the gradient (gradient_(sample)) was determined.

40 μL of the control reaction solution was added to 10 μL each of anaqueous glucose solution at a concentration of 125 mg/dL, an aqueousglucose solution at a concentration of 250 mg/dL, and an aqueous glucosesolution at a concentration of 1,000 mg/dL, and the mixtures wereallowed to develop color. The spectra were measured with aspectrophotometer (measurement cell length: 50 μm) (n=1). For each ofthe spectra, the absorbance at 650 nm, which is the measurementwavelength (λmax) of WST-4, was measured, and the glucose concentrationin the colored solution and the absorbance were plotted on thehorizontal axis and the vertical axis, respectively. Thus, the gradient(gradient_(WST-4)) was determined.

In a case in which the value obtained by dividing the gradient_(sample)determined as described above by the gradient(gradient_(sample)/gradient_(WST-4)) was 2 or greater, the sensitivitywas rated as “

”; in a case in which the value of the ratiogradient_(sample)/gradient_(wsT-4) was 1.5 or greater and less than 2,the sensitivity was rated as “◯”; in a case in which the value of theratio gradient_(sample)/gradient_(wsT-4) was greater than 1 and lessthan 1.5, the sensitivity was rated as “Δ”; and in a case in which thevalue of the ratio gradient_(sample)/gradient_(WST-4) was 1 or less, thesensitivity was rated as “×”. As an example, the plot diagrams for thetetrazolium compound 1 and WST-4 are presented in FIG. 2. FIG. 2 is agraph showing the relationship between the glucose concentration and theabsorbance of produced formazan in relation to the tetrazolium compound1 and WST-4. From FIG. 2, it was found that since the gradients of thecompound 1 and WST-4 were 0.0063 and 0.0026, respectively, the ratiogradient_(sample)/gradient_(WST-4) was about 2.4. Meanwhile, thegradient serves as an index for the color development intensity of eachcompound. Therefore, when the value obtained by dividing thegradient_(sample) by the gradient_(WST-4)(gradient_(sample)/gradient_(WST-4)) is greater than one-fold, it isimplied that the intensity of color development on the longer wavelengthside is higher compared to WST-4, which is a well-known indicatorreagent.

(Evaluation of Water-Solubility)

Each compound was added to 100 μL of RO water and 100 μL of a 0.5 M MOPSsolution (pH 7.2), in an amount such that the concentration of thecompound in the aqueous solution would be 200 mM. After the mixture wasstirred for 5 minutes, a precipitate was checked by visual inspection.Separately, each compound was added to 100 μL of RO water and 100 μL ofa 0.5 M MOPS solution (pH 7.2), in an amount such that the concentrationof the compound in the aqueous solution would be 100 mM. After themixture was stirred for 5 minutes, a precipitate was checked by visualinspection. Separately, each compound was added to 100 μL of RO waterand 100 μL of a 0.5 M MOPS solution (pH 7.2), in an amount such that theconcentration of the compound in the aqueous solution would be 20 mM.After the mixture was stirred for 5 minutes, a precipitate was checkedby visual inspection. In a case in which there was no precipitate in the200 mM aqueous solution, the solubility was rated as

. In a case in which a precipitate was seen in the 200 mM aqueoussolution, but there was no precipitate in the 100 mM aqueous solution,the solubility was rated as ◯. In a case in which a precipitate was seenin the 100 mM aqueous solution, but there was not precipitate in the 20mM aqueous solution, the solubility was rated as “Δ”. In a case in whicha precipitate was recognized even in the 20 mM aqueous solution, thesolubility was rated as “×”.

(Evaluation of Stability)

To 0.02 mmol of each compound, 75 μL of RO water and 100 μL of a 0.5 MMOPS solution (pH 7.2) were added, and the compound was dissolvedtherein. The solution was used as a sample.

To 175 μL of this sample, 10 μL of the GDH solution, 5 μL of the m-PMSsolution, and 10 μL of the nickel solution, all of which had beenproduced in the same manner as in the above section (Evaluation ofsensitivity), were added, and thereby a measurement solution wasproduced.

As a control, 85 μL of RO water and 100 μL of a 0.5 M MOPS solution (pH7.2) were added to 11.6 mg of2-benzothiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylcarbamoyl)phenyl]-2H-tetrazolium(WST-4), and the compound was dissolved. Thus, a control sample wasproduced. To 175 μL of the control sample, 10 μL of the GDH solution and5 μL of the m-PMS solution, both of which had been produced as describedabove, and 10 μL of RO water were added. Thus, a control reactionsolution was produced.

At the time of production of the measurement solution (0 hour), andafter 1 hour, 2 hours, and 6 hours from the production, the spectra ofthe measurement solutions produced as described above were measuredusing a spectrophotometer (measurement cell length: 50 μm) (n=1). Foreach spectrum, the absorbance at the maximum absorption wavelength(λmax) of each compound as determined above was measured. Theabsorbances measured at the time of production of the measurementsolution (0 hour) and after 6 hours from the production were designatedas Abs_(0 h) and Abs_(6 h), respectively. In a case in which the valueobtained by subtracting the absorbance at the time of production of themeasurement solution (0 hour) from the absorbance after 6 hours anddividing the resultant by the measurement time [(Abs_(6 h) Abs_(0 h))/6]was 0.01 or less, the stability was rated as “

”; in a case in which the above-mentioned value was more than 0.01 and0.05 or less, the stability was rated as “◯”; in a case in which theabove-mentioned value was more than 0.05 and 0.1 or less, the stabilitywas rated as “Δ”; and in a case in which the above-mentioned value wasmore than 0.1, the stability was rated as “×”. For example, thestability evaluation results for tetrazolium compound 1 are presented inFIG. 3. In FIG. 3, the absorbances after 1 hour, after 2 hours, andafter 24 hours from the production of the measurement solution are alsoshown. From FIG. 3, it is understood that since the[(Abs_(6 h)-Abs_(0 h))/6] value of the compound 1 was about 0.008, thestability was

. Meanwhile, the [(Abs_(6 h)−Abs_(0 h))/6] value of the control reactionsolution was about 0.01.

TABLE 3 Substituted sulfonated phenyl group existing at 5-positionSubstituted sulfonated phenyl group existing at Sensitivity Substitutedbenzothiazolyl of tetrazole skeleton 3-position of tetrazole skeletonMaximum Comparison group existing at 2-position Position Position ofabsorption with of tetrazole skeleton of sulfuric Position sulfuric acidwavelength Chelation Comparative Water- OR³ q R¹ m acid group R² n of R²p group (λmax) (nm) rate Example 5 solubility Stability Example 1Compound 1 6-Methoxy group 1 Hydrogen 2 2,4-position Methoxy group 14-position 1 3-position 630 ◯ ⊙ ⊙ ⊙ Example 2 Compound 2 6-Methoxy group1 Hydrogen 2 2,4-position Methoxy group 1 2-position 1 5-position 600 ◯⊙ ⊙ ⊙ Example 3 Compound 3 6-Methoxy group 1 Hydrogen 2 2,4-positionMethoxy group, 2 2-position, 1 5-position 630 ◯ ◯ ⊙ ◯ Nitro group4-position Example 4 Compound 4 6-Methoxy group 1 Hydrogen 22,4-position Methoxy group, 2 2-position, 0 X 650 ◯ ⊙ Δ ⊙ Nitro group4-position Example 5 Compound 5 6-Methoxy group 1 Hydroxyl 1 2-positionMethoxy group 1 2-position 1 5-position 630 ◯ ◯ ◯ ⊙ group Example 6Compound 6 6-Methoxy group 1 Hydroxyl 1 3-position Methoxy group 12-position 1 5-position 650 ◯ ⊙ Δ ⊙ group Example 7 Compound 7 6-Methoxygroup 1 Hydrogen 1 2-position — 0 — 1 4-position 645 ◯ ⊙ ◯ Δ Example 8Compound 8 6-Methoxy group 1 Hydrogen 2 3,5-position Methoxy group 12-position 1 5-position 620 ◯ ◯ ⊙ ⊙ Example 9 Compound 9 6-Methoxy group1 Hydrogen 2 3,5-position Methoxy group 1 4-position 1 3-position 640 ◯◯ ⊙ ⊙ Example 10 Compound 10 6-Methoxy group 1 Hydrogen 2 2,4-positionMethoxy group, 2 2-position, 0 X 620 ◯ ◯ ⊙ ⊙ Carboxy group 4-positionExample 11 Compound 11 6-Methoxy group 1 Hydrogen 2 3,5-position Methoxygroup, 2 2-position, 0 X 605 ◯ ◯ ⊙ ⊙ carboxy group 5-position Example 12Compound 12 6-Methoxy group 1 Hydrogen 2 2,4-position Carboxy group, 23-position, 0 X 630 ◯ ⊙ 2.4 times ⊙ ⊙ methoxy group 4-position Example13 Compound 13 5,6-Methylenedioxy- 2 Hydrogen 2 2,4-position Methoxygroup 1 2-position 1 5-position 625 ◯ ⊙ 2.4 times ⊙ ⊙ 1,3-benzothiazoleExample 14 Compound 14 6,7-dihydro[1,4]dioxino[2,3- 2 Hydrogen 22,4-position Methoxy group 1 2-position 1 5-position 610 ◯ ⊙ 2.4 times ⊙⊙ f][1,3]benzothiazole Example 15 Compound 15 6-Ethoxy group 1 Hydrogen2 2,4-position Methoxy group 1 2-position 1 5-position 605 ◯ ⊙ 2.4 times⊙ ⊙ Example 16 Compound 16 5,6-Dimethoxy 2 Hydrogen 2 2,4-positionMethoxy group 1 2-position 1 5-position 625 ◯ ⊙ 2.4 times ⊙ ⊙ Example 17Compound 17 5,6-Methylenedioxy- 2 Hydrogen 2 2,4-position Methoxy group1 4-position 1 5-position 650 ◯ ⊙ 2.4 times ⊙ ⊙ 1,3-benzothiazoleExample 18 Compound 18 5,6-Dimethoxy 2 Hydrogen 2 2,4-position Methoxygroup 1 4-position 1 5-position 650 ◯ ⊙ 2.4 times ⊙ ⊙ Example 19Compound 19 6,7-dihydro[1,4]dioxino[2,3- 2 Hydrogen 2 2,4-positionMethoxy group 1 4-position 1 5-position 640 ◯ ⊙ 2.4 times ⊙ ⊙f][1,3]benzothiazole Example 20 Compound 20 6-Methoxy group 1 Hydrogen 22,4-position Methoxy group 1 4-position 1 5-position 630 ◯ ⊙ 2.4 times ⊙⊙ Comparative Comparative — — Hydrogen 2 2,4-position Methoxy group 12-position 1 5-position 580 ◯ Δ ◯ ◯ Example 1 Compound 1 ComparativeComparative Nitro group — Hydrogen 2 2,4-position Methoxy group 12-position 1 5-position No chelation X — — — Example 2 Compound 2Comparative Comparative — — Hydrogen 2 2,4-position Methyl group 12-position 1 4-position 560 ◯ ⊙ ◯ ◯ Example 3 Compound 3 ComparativeComparative — — Hydrogen 2 2,4-position Methoxy group 1 2-position 0 —580 ◯ ⊙ ◯ ◯ Example 4 Compound 4 Comparative Comparative — —2-Sulfoethyl- 0 Methoxy group, 2 — 0 — No chelation X 1 ◯ ◯ Example 5Compound 5 carbamoyl carboxyl group group

From the results of Table 3 shown above, it can be seen that the bloodsugar level can be measured rapidly with satisfactory sensitivity byusing the tetrazolium salts of the Examples.

Furthermore, the chelate compounds of the formazans produced from thetetrazolium salts of the Examples and nickel ion exhibit maximumabsorption wavelengths (λmax) of 600 nm or higher. Therefore, it iscontemplated that even for a whole blood sample, the biologicalcomponent concentration such as the blood sugar level can be measuredaccurately with high sensitivity.

As shown in Table 3, the tetrazolium salts of the present disclosureexhibit satisfactory water-solubility. From the results given above, itis speculated that the formazans produced from the tetrazolium salts ofthe present disclosure, and chelate compounds of those formazans andtransition metal ions also exhibit satisfactory water-solubility,similarly to the tetrazolium salts of the present disclosure.

In addition, tetrazolium salts 1 to 3, 5, and 7 to 20, each satisfyingany one of the following conditions: (1) m=2 and p=1; (2) m=1 and n=0;or (3) R¹ represents a hydroxyl group, and at this time, the sulfo group(SO³⁻) and the hydroxyl group are at the 2,4-position or at the 4- and6-positions; or (4) p=0 and at least one of R²'s represents a carboxylgroup, exhibit more satisfactory water-solubility. Examples 1 to 3 and 8to 20, in which (1) m=2 and p=1, or (4) p=0 and at least one of R²'srepresents a carboxyl group, exhibit particularly satisfactorywater-solubility.

Meanwhile, the compound of Comparative Example 2, which is a tetrazoliumsalt obtained by changing the alkoxy group of the benzothiazolyl groupof Example 2 into an electron-withdrawing nitro group, does not form achelate between formazan and a transition metal ion. Therefore, it isunderstood that substituting the benzothiazolyl group existing at the2-position of the tetrazole ring with an alkoxy group is very importantfor shifting the maximum absorption wavelength toward the longerwavelength side while maintaining the ability of formazan produced fromthe tetrazolium salt to forma chelate with a transition metal compound.

Evaluation Example 1: Evaluation as Blood Glucose Meter Sensor

1. Production of Coating Liquid

75 μL of RO water and 100 μL of a 0.5 M MOPS solution (pH 7.2) wereadded to 14.5 mg of tetrazolium compound 1 obtained in Example 1 todissolve the tetrazolium compound therein, and thus a sample wasproduced. As a control, 85 μL of RO water and 100 μL of a 0.5 M MOPSsolution (pH 7.2) were added to 11.6 mg of2-benzothiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylcarbamoyl)phenyl]-2H-tetrazolium(WST-4) to dissolve the compound therein, and thus a control sample wasproduced.

Separately, 100 μL of RO water was added to 7 mg of a glucosedehydrogenase (GDH-FAD) (manufacturedbyTOYOBOCO., LTD., product No.:GLD-351) to dissolve the enzyme, and thus a GDH solution was produced.Furthermore, 200 μL of RO water was added to 3.5 mg of1-methoxy-5-methylphenazium methyl sulfate (m-PMS) (manufactured byDOJINDO LABORATORIES) to dissolve the compound, and thus an s-PMSsolution was produced. 1 mL of RO water was added to 129 mg of nickelchloride to dissolve the salt, and thus a nickel solution was produced.

To the sample, 10 μL of the GDH solution, 5 μL of the s-PMS solution,and 10 μL of the nickel solution, all of which had been produced asdescribed above, were added, and thereby a coating liquid was produced.Furthermore, 10 μL of the GDH solution and 5 μL of the s-PMS solutionproduced as described above were added to the control sample, andthereby a control coating liquid was produced.

2. Production of Reagent Ribbon

The coating liquid obtained as described above was applied on a PETfilm, and a reagent ribbon 1 was produced. The area of coating was 1.5mm×75 mm (total coating amount 5.6 μL). This reagent ribbon 1 was cut inthe longitudinal direction, and a reagent piece 2 was obtained (1.5 mm×3mm).

3. Assembly of Blood Glucose Meter Sensor

Along the two edges of a PET film 5 that would become one surface of ablood glucose meter sensor, a double-sided tape having a thickness of 50μm was provided as a spacer. Next, the reagent-coated surface of thereagent piece 2 was placed to face downward, and the reagent piece 2 wasmounted on the double-sided tape 4. Thus, a blood glucose meter sensorwas assembled. FIG. 4 is a schematic diagram illustrating the assemblyof a blood glucose meter sensor. In FIG. 4, a reagent piece 2 isobtained by cutting the reagent ribbon 1, and then with thereagent-coated surface 3 of the reagent piece 2 being arranged to facedownward, the reagent piece 2 is installed on the double-sided tape 4disposed at the two edges of the PET film 5. Subsequently, a PET film 7is installed at the remaining adhesive parts of the double-sided tape asshown in the diagram. At this time, a flow channel 6 is formed betweenthe double-sided tapes.

A whole blood sample (Ht40, 100 mg/dL) (hematocrit value 40%, glucoseconcentration 100 mg/dL) was spotted at the flow channel inlet port ofthe blood glucose meter sensor thus produced. The whole blood sample(Ht40, 100 mg/dL) was produced as follows: whole blood was collectedinto a heparin-containing blood sampling tube, the hematocrit value ofthis whole blood was measured, and the hematocrit value of the wholeblood sample was regulated to be Ht40 by adding the blood plasmaobtained by separation in advance, or by removing the blood plasma ofthe whole blood sample, as appropriate. Furthermore, a highlyconcentrated glucose solution (40 g/dL) was added as appropriate to thiswhole blood sample, and a whole blood sample (Ht40, 100 mg/dL) wasproduced. The specimen was spotted on the reagent part, and then after 9seconds, the spectrum was measured using a fiber spectrophotometer. Theabsorbance at the maximum absorption wavelength was measured using thespectrum measured with each of the blood glucose meter sensors that usedvarious compounds. The absorbance obtainable in the case of measuringthe whole blood sample (Ht40, 100 mg/dL) was designated as Abs_(BG100).

Furthermore, a spectrum was similarly measured using a specimen having ablood sugar level of 0 mg/dL (Ht40, 0 mg/dL) as a control, and theabsorbance at the maximum absorption wavelength with each of variouscompounds (compound 1 and WST-4) was designated as Abs_(BG0). Here, thespecimen having a blood sugar level of 0 mg/dL (Ht40, 0 mg/dL) wasproduced as follows. To 1 mL of the whole blood sample (Ht40, 100mg/dL), 0.1 mg of a glucose oxidase (GLO-201 manufactured by TOYOBO CO.,LTD.) was added. After the addition, the mixture was left to stand atroom temperature for 5 minutes, and thus a whole blood sample (Ht40, 0mg/dL) was produced.

The results of determining the values of ΔAbs=Abs_(BG100)−Abs_(BG0) ofvarious compounds are presented in FIG. 5.

As shown in FIG. 5, in the blood glucose meter sensor to whichtetrazolium compound 1 of Example 1 was applied, color developmentoccurred to an extent of about 3 times the color development of WST-4(650 nm). Therefore, it can be seen that the reagent for biologicalcomponent concentration measurement including the tetrazolium salt ofthe present disclosure can detect a biological component with highsensitivity, even in the case of using a whole blood sample.

Furthermore, a whole blood sample (Ht40, 400 mg/dL) (hematocrit value40%, glucose concentration 400 mg/dL) was produced similarly to thewhole blood sample (Ht40, 100 mg/dL). Separately, an aqueous glucosesolution (100 mg/dL) and an aqueous glucose solution (400 mg/dL) wereproduced. For these samples, spectra were measured with aspectrophotometer after 9 seconds from the initiation of reaction in thesame manner, using a blood glucose meter sensor to which thetetrazolium. compound 1 obtained in Example 1 was applied. The resultsare shown in FIG. 6. Meanwhile, FIG. 6A is a diagram showing the spectraobtained when the whole blood samples were submitted to the bloodglucose meter sensor to which the tetrazolium compound 1 was applied.FIG. 6B is a diagram showing the spectra obtained when aqueous glucosesolutions were submitted to the blood glucose meter sensor to which thetetrazolium compound 1 was applied.

As shown in FIG. 6, with the blood glucose meter sensor to which thetetrazolium compound 1 of Example 1 was applied, the absorbances of boththe whole blood and the aqueous glucose solutions were higher than 0.2.Therefore, it can be seen that even in the case of using whole bloodsamples, the noise caused by colored components among the bloodcomponents is low, and biological components can be detected with highsensitivity.

Furthermore, an aqueous glucose solution (800 mg/dL) was produced.

Each sample of the whole blood sample (Ht40, 0 mg/dL), the whole bloodsample (Ht40, 100 mg/dL), the whole blood sample (Ht40, 400 mg/dL), theaqueous glucose solution (100 mg/dL), the aqueous glucose solution (400mg/dL), and the aqueous glucose solution (800 mg/dL) thus produced wasmeasured with a blood glucose meter sensor to which the tetrazoliumcompound 1 of Example 1 was applied, and the relationship between thetime taken from the initiation of reaction and the absorbance at 630 nmwas determined. The results are shown in FIG. 7. FIG. 7A is a diagramshowing the relationship between the time taken from the initiation ofreaction when each of the whole blood samples was spotted on the bloodglucose meter sensor to which the tetrazolium compound 1 was applied,and the absorbance at 630 nm. FIG. 7B is a diagram showing therelationship between the time taken from the initiation of reaction wheneach of the glucose solutions was submitted to the blood glucose metersensor to which the tetrazolium compound 1 was applied, and theabsorbance at 630 nm.

As shown in FIG. 7, in the blood glucose meter sensor to which thetetrazolium compound 1 of Example 1, color development was completed in5 seconds for both the whole blood and the aqueous glucose solution, andthe rate of color development and sensitivity were all satisfactory.Therefore, the reagent for biological component concentrationmeasurement including the tetrazolium salt of the present disclosure candetect biological components rapidly with high sensitivity, even in thecase of using whole blood samples.

Furthermore, FIG. 8 presents a graph showing the relationship betweenthe glucose concentration and the absorbance at the maximum absorptionwavelength of a chelate compound of formazan and Ni²⁺ when the wholeblood sample (Ht40) and aqueous glucose solutions were measured with ablood glucose meter sensor. The measurement of the absorbance wascarried out after 9 seconds from the initiation of reaction. FIG. 8A isa graph showing the relationship between the glucose concentration andthe absorbance of a chelate compound of formazan and Ni²⁺ when a wholeblood sample was submitted to the blood glucose meter sensor to whichthe tetrazolium compound 1 was applied. FIG. 8B is a graph showing therelationship between the glucose concentration and the absorbance of achelate compound of formazan and Ni²⁺ when an aqueous glucose solutionwas submitted to the blood glucose meter sensor to which the tetrazoliumcompound 1 was applied.

As shown in FIG. 8, the absorbance and the blood sugar level exhibit alinear relationship (proportional relationship). From this, it iscontemplated that the blood sugar level can be measured accurately bymeans of the absorbance.

From these findings, when the tetrazolium salt of the presentdisclosure, an oxidoreductase, and a transition metal compound are addedto a biological sample, the quantity of color development is measured,and a calibration curve is produced based on this quantity of colordevelopment, the concentration of a biological component in a biologicalsample can be calculated accurately.

Evaluation Example 2: Evaluation with Blood Glucose Meter Sensor

1. Production of Ni Acetate and DSB 20 mM Solution

0.6 mL of a 0.5 M Ni acetate solution, 0.4 mL of RO water, and 8.7 mg ofDSB (benzene-1,3-disulfonic acid) were mixed, and a Ni acetate-DSBsolution was produced.

2. Production of Coating Liquid

To 87 μL of the Ni acetate-DSB solution, 4.7 μL of a 1 N NaOH solution,30.3 μL of RO water, and 4.8 mg of each of the tetrazolium salts(compounds 17 to 20 and compound 1) were added, and the mixture wasmixed. Furthermore, 4 μL of methanol and 1.7 mg of GDH-FAD were added tothis liquid. This liquid was centrifuged, and the supernatant was usedas a coating liquid.

3. Production of Reagent Piece

The coating liquid obtained as described above was applied on a PET filmby an inkjet method, and a reagent ribbon was produced. This reagentribbon was cut in the longitudinal direction (1.5 mm×3 mm), and this wasdesignated as reagent piece 2.

4. Assembly of Blood Glucose Meter Sensor

FIG. 13A is a schematic diagram illustrating the assembly of a bloodglucose meter sensor. In FIG. 13A, after the reagent piece 2 wasobtained, a reagent-coated surface 3 of the reagent piece 2 was arrangedto face downward, and the reagent piece 2 was installed on a PET film 5.Thus, a blood glucose meter sensor was assembled. In FIG. 13A, thereagent-coated surface 3 of the reagent piece 2 was arranged to facedownward, and the reagent piece 2 was installed on double-sided tapes 4disposed as spacers along the two edges of the PET film 5. Subsequently,a PET film 7 having the same shape as that of the PET film 5 was furtherattached by pressing on the reagent piece 2, and thereby, a sensor chipin which the blood flow channel at the inner surface had a stepped shapewas obtained. FIG. 13B is a cross-sectional view in the longitudinaldirection or the transverse direction of the sensor chip. Here, in FIG.13B, regarding the flow channel length (L1), the flow channel width (W),and the flow channel thickness (t1) of the flow channel part, and theflow channel length (L2), the flow channel width (W), and the flowchannel thickness (t2) of the reagent part (measuring unit)corresponding to a reagent-coated part, the dimensions indicated in thefollowing table were used. In FIG. 13B, the flow channel of the chip iscomposed of a reagent part formed by a reagent-coated surface disposedthereon; and a flow channel part formed from the portion of flow channelwithout the reagent-coated surface disposed thereon in the flow channel.

TABLE 4 Flow channel part Reagent part (measuring unit) Length L1 9 mmLength L2 3 mm Width W 1.5 mm Width W 1.5 mm Thickness t1 0.13 mmThickness t2 0.05 mm

The length L1 in a direction orthogonally intersecting the chipthickness direction (flow channel longitudinal direction) in the flowchannel part is not particularly limited and can be selected asappropriate according to the purpose; however, the length L1 ispreferably 5 to 10 mm. Here, when the length L1 is long, it isadvantageous from the viewpoint that mounting (insertion) into acomponent analyzer is easy, and from the viewpoint that the intrusion ofambient light into the photometric part is reduced. When the length L1is short, it is advantageous from the viewpoint that the amount ofspecimen can be reduced. Therefore, in the light of the balance betweenthe ease of mounting (insertion) into a component analyzer, theinfluence of ambient light, and the amount of specimen, the upper limitand the lower limit of the length L1 are determined. The length L2 in adirection orthogonally intersecting the chip thickness direction (flowchannel longitudinal direction) of the flow channel in the reagent partis not particularly limited and can be selected as appropriate accordingto the purpose; however, the length L2 is preferably 1 to 4 mm. Here,when the length L2 is long, it is advantageous from the viewpoint thatsince the area of the irradiation spot is taken to be large in thelength direction, measurement can be carried out very accurately.Meanwhile, when the length L2 is short, it is advantageous from theviewpoint that the amount of specimen can be reduced. Therefore, in thelight of the balance between the measurement accuracy and the amount ofspecimen, the upper limit and the lower limit of the length L2 aredetermined.

4. Evaluation of Produced Sensor

Aqueous glucose solution (n=3) or whole blood (n=5) was spotted on asensor including each of the tetrazolium salts thus produced, and theblank spectrum, the color development spectrum, and the colordevelopment time course were checked using a fiber spectrophotometer. Asthe absorbance of the indicator compound, the absorbance at 650 nm wasmeasured.

FIG. 14 presents the blank spectrum obtained at the time of spottingwater having a glucose concentration of 0 mg/dL on the sensor, and FIG.15 presents a differential spectrum obtained by subtracting theabsorbance spectrum obtained by spotting water having a glucoseconcentration of 0 mg/dL (FIG. 14), from the absorbance spectrum of thesensor on which aqueous glucose solution having a glucose concentrationof 400 mg/dL was spotted (the differential spectrum means the netquantity of color development of glucose).

In regard to the tetrazolium compound of the present disclosure, thetime course of the net quantity of color development (measurementwavelength 650 nm) was measured for various substituents of the alkoxygroup substituted at the 6-position (or the 5,6-position) of thebenzothiazole ring. Asa compensating wavelength that reflects the noisecomponent originating from the blood sample, the absorbance at 900 nmwas also measured. The time course of the quantity of color developmentwas described as the proportion % at various times obtained when theabsorbance after 15 seconds from the initiation of reaction was denotedas 100% with regard to the measurement wavelength 650 nm. The “netquantity of color development” is the net quantity of color developmentobtained by subtracting the absorbance obtained when blood having ablood sugar level of 0 mg/dL was reacted with the blood sugar levelmeasuring reagent, from the absorbance obtained when blood having adesired blood sugar level (for example, 800 mg/mL) was reacted with theblood sugar level measuring reagent, with regard to blood samples havingthe same hematocrit value. Regarding the absorbances used for thecalculation of the quantity of color development, a value obtained bysubtracting the absorbance at 900 nm for compensating opticalfluctuations, from the absorbance at 650 nm was used, in order toeliminate any optical fluctuations other than color development. Theabsorbance at 650 nm includes noise caused by the quantity of colordevelopment originating from glucose and the scattered light originatingfrom blood cells. The absorbance at 900 nm reflects the quantity of thenoise at 650 nm caused by the scattered light. FIG. 16 presents theabsorbance spectrum obtainable when an aqueous glucose solution (800mg/dL) was used, and FIG. 17 presents the absorbance spectrum obtainablewhen a whole blood sample (Ht40, 800 mg/dL) (hematocrit value 40%,glucose concentration 800 mg/dL) was used. From these results, it wasconfirmed that compound 17, compound 18, compound 19, and compound 20,in which the benzothiazole ring of compound 1 was substituted withvarious alkoxy groups at the 6-position or at the 5,6-position, can allbe used as reagents for glucose measurement. Meanwhile, in EvaluationExamples 1 and 2, evaluation was made in consideration of the influenceof the cell length in the measurement of transmitted light.

Evaluation Example 3: Estimation of Molar Extinction Coefficient

A measurement solution was produced in the same manner as described inthe section for the evaluation of the chelation rate and the maximumabsorption wavelength (λmax), using the tetrazolium compound 1 ofExample 1.

Estimation of the molar extinction coefficient was carried out using theblood glucose meter sensor of FIG. 13. At this time, it is assumed thattetrazolium compound 1 reacts with glucose at a ratio of 1:1 (molarratio). The concentration (mol/L) of β-glucose include in the spottedaqueous glucose solution was designated as x, and the value obtained bycalculating the absorbance at 630 nm of the blood glucose meter sensorhaving aqueous glucose solution spotted thereon, to a value per 1 cm,was designated as y. The gradient of a straight line obtained at thattime is designated as the molar extinction coefficient (calculated basedon a glucose molecular weight 180.16, and a β-glucose ratio in thesolution of 62%).

FIG. 18 presents a graph obtained when the concentration (mol/L) ofβ-glucose was used as x value, and the absorbance at λmax=635 nm ofcolored chelate compound of formazan and Ni²⁺, which was calculated intoa value per 1 cm (abs/cm), was used as y value. From the graph, themolar extinction coefficient at λmax=635 nm of a colored chelatecompound of formazan and Ni²⁺ is c=22,594 L/mol·cm.

REFERENCE SIGNS LIST

1 REAGENT RIBBON; 2 REAGENT PIECE; 3 REAGENT-COATED

SURFACE; 4 DOUBLE-SIDED TAPE; 5 PET FILM; 6 FLOW CHANNEL; 7 PET FILM; 10BLOOD GLUCOSE METER; 12 SENSOR CHIP; 14 MEASURING UNIT; 16 APPARATUSMAIN BODY; 18 CHIP MAIN BODY; 20 CAVITY; 20 a FRONT END PORT; 20 b BASEEND PORT; 22 LONG EDGE; 22 a UPPER LONG EDGE; 22 b LOWER LONG EDGE; 24SHORT EDGE; 24 a FRONT END EDGE; 24 b BASE END EDGE; 26 REAGENT; 28MEASURING OBJECT PORTION; 30 PLATE PIECE; 32 SPACER; 40 CASE; 42 CONTROLUNIT; 44 BOX BODY; 46 PHOTOMETRIC UNIT; 48 POWER SUPPLY BUTTON; 50OPERATION BUTTON; 52 DISPLAY; 54 EJECTION LEVER; 56 EJECTION PIN; 56 aROD PORTION; 56 b RECEPTOR; 58 INSERTION PORT; 58 a INSERTION OPENING;59 PORT FOR MEASUREMENT; 60 CHIP MOUNTING PART; 60 a FLANGE PORTION; 62WALL; 64 DEVICE ACCOMMODATING SPACE; 66 LIGHT GUIDE; 68 LIGHT EMITTINGDEVICE; 70 LIGHT EMITTING UNIT; 72 LIGHT RECEIVING DEVICE; 74 LIGHTRECEIVING UNIT; 76 COIL SPRING.

What is claimed is:
 1. A 2-substituted benzothiazolyl-3-substitutedphenyl-5-substituted sulfonated phenyl-2H-tetrazolium salt representedby the following Formula (1):

wherein in Formula (1), R¹ represents any one selected from the groupconsisting of a hydrogen atom, a hydroxyl group, a methoxy group, and anethoxy group; R² represents any one selected from the group consistingof a nitro group, —OR⁴, and a carboxyl group; R³ represents a hydrogenatom, a methyl group, or an ethyl group, while at least one is a methylgroup or an ethyl group; R⁴ represents a methyl group or an ethyl group;m represents the number of sulfo groups (—SO₃ ⁻) bonded to the phenylgroup at the 5-position of the tetrazole skeleton, and is 1 or 2; nrepresents the number of R² bonded to the phenyl group at the 3-positionof the tetrazole skeleton, and is an integer from 0 to 2; p representsthe number of sulfo groups (—SO₃ ⁻) bonded to the phenyl group at the3-position of the tetrazole skeleton, and is 0 or 1; n+p is 1 orgreater; q is 1 or 2; when q is 2, the OR³'s are disposed adjacently toeach other and may be bonded to each other and forma ring; and Xrepresents a hydrogen atom or an alkali metal atom.
 2. The tetrazoliumsalt of claim 1, wherein in Formula (1), m is
 2. 3. The tetrazolium saltof claim 1, wherein in Formula (1), p is 1, or p is 0, with at least oneR² is a carboxyl group.
 4. The tetrazolium salt of claim 1, wherein inFormula (1), the phenyl group at the 5-position of the tetrazoleskeleton is a phenyl group having a sulfo group (—SO₃ ⁻) at the2-position or the 4-position.
 5. The tetrazolium salt of claim 1,wherein in Formula (1), at least one —OR³ of the substitutedbenzothiazolyl group existing at the 2-position of the tetrazoleskeleton is bonded to the 6-position of the benzothiazolyl group.
 6. Thetetrazolium salt of claim 1, wherein in Formula (1), n is 1 or 2, and atleast one R² is an —OR⁴ group.
 7. The tetrazolium salt of claim 6,wherein the —OR⁴ group is a methoxy group.
 8. The tetrazolium salt ofclaim 1, wherein in Formula (1), p is 1, and the phenyl group at the3-position of the tetrazole skeleton is a phenyl group having a sulfogroup (—SO₃ ⁻) existing at the 3-position or the 5-position.
 9. Thetetrazolium salt of claim 1, wherein in Formula (1), the phenyl groupexisting at the 3-position of the tetrazole skeleton is a4-methoxy-3-sulfophenyl group, a 2-methoxy-5-sulfophenyl group, a3-carboxy-4-methoxyphenyl group, or a 4-methoxy-5-sulfophenyl group. 10.A reagent for biological component concentration measurement, thereagent comprising the tetrazolium salt of claim
 1. 11. The reagent forbiological component concentration measurement of claim 10, the reagentfurther comprising a transition metal compound.
 12. The reagent forbiological component concentration measurement of claim 11, wherein thetransition metal compound is a nickel compound.
 13. The reagent forbiological component concentration measurement of claim 10, wherein thereagent is used for the measurement of the concentration of glucose,cholesterol, neutral lipids, nicotinamide adenine dinucleotide phosphate(NADPH), nicotinamide adenine dinucleotide (NADH), or uric acid in bloodor a body fluid.
 14. A method for measuring a concentration of abiological component, the method comprising: adding the 2-substitutedbenzothiazolyl-3-substituted phenyl-5-substituted sulfonatedphenyl-2H-tetrazolium salt of claim 1, an oxidoreductase, and atransition metal compound to a biological sample; measuring the quantityof color development; and quantitatively determining the concentrationof a biological component in the biological sample based on the quantityof color development.
 15. The method of claim 14, wherein the biologicalcomponent in the biological sample is glucose, cholesterol, neutrallipids, nicotinamide adenine dinucleotide phosphate (NADPH),nicotinamide adenine dinucleotide (NADH), or uric acid in blood or abody fluid.
 16. A blood sugar level measuring reagent, comprising a2-substituted benzothiazolyl-3-substituted phenyl-5-substitutedsulfonated phenyl-2H-tetrazolium salt, GDH-FAD, and a transition metalion.
 17. A method for measuring blood sugar level, the method comprisingadding a reagent of a 2-substituted benzothiazolyl-3-substitutedphenyl-5-substituted sulfonated phenyl-2H-tetrazolium salt, GDH-FAD, anda transition metal ion to a sample.